november9san diego poster correctio october 26
TRANSCRIPT
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HIV induces complement C3 in primary human astrocytes indirectly through a NFkB-dependent pathwayJadwiga Nitkiewicz, Wei Chao, Seon-Young Kim, Luni Nemdad, Devanand Sarkar, Paul Fisher and David J. VolskyMolecular Virology Division, St. Lukes-Roosevelt Hospital Center, and Department of Pathology Columbia University Medical Center, New York, NY 10019INTRODUCTIONInnate immunity is believed to play an important role in protecting brain against HIV infection and development of HIV-associated dementia (HAD). Complement C3 is a key component of the innate immunity system but in models of other neurodegenerative diseases such as multiple sclerosis, uncontrolled C3 production can lead to tissue injury and inflammation. C3 is secreted by several cell types in the brain including astrocytes. We and others have previously shown that HIV interaction with astrocytes in vitro alters cellular gene expression, induces interleukin-6, and disrupts important physiological functions of the cells such as glutamate transport. Here we investigated the mechanism of C3 induction in human fetal astrocytes (HFA) exposed to HIV in vitro. Using transient transfection assays with a C3 promoter-driven indicator gene, real-time PCR, and C3 ELISA we found that exposure of astrocytes to HIV increased C3 promoter activity, mRNA expression, and protein production, indicating that HIV acts by transcriptional activation of the C3 gene. Studies with pharmacological inhibitors revealed that activation of C3 by HIV requires, among others, the NFkB and PTK signaling pathways; however, surprisingly, the C3 core promoter lacks NFkB and SP binding sites. Activation of the C3 promoter by HIV was absolutely dependent on the presence of the interleukin 1-beta/interleukin 6 responsive element at -109 to -90 and its sub-domain -106 to -100. The requirement for functional NFkB for HIV effect on C3 was confirmed by overexpresion of the NFkB inhibitor IkB-a in astrocytes; adenovirus-mediated transduction of IkB-a into HFA, but not of control virus, blocked nuclear translocation of NFkB and prevented induction of C3 promoter and mRNA. FIGURE 2A, BEFFECT OF PHARMACOLOGICAL INHIBITORS OF SIGNAL TRANSDUCTION PATHWAY ON C3 PROMOTER INDUCTION BY HIV IN ASTROCYTESFIGURE 2C EFFECT OF INHIBITORS OF NFKAPPA B (CAPE) and PTK (GENISTEIN) ON C3 GENE EXPRESSION AFTER HIV EXPOSURE OF PRIMARY ASTROCYTESFIGURE 1 A. DETECTION OF C3 PROTEIN SECRETION IN CELL SUPERNATANTS B. C3 GENE EXPRESSION IN EARLY (24H), AND LATE (7 D) TIMEPOINT TESTED IN CELL EXTRACTC. INDUCTION OF C3 PROMOTER AFTER 48H; TESTED IN CELL EXTRACTFIGURE 3A DETECTION OF p50 SUBUNITS OF ACTIVATED NFkappaBIN NUCLEAR EXTRACT (AFTER 30 MIN OF THE HIV EXPOSURE OF PRIMARY ASTROCYTES BY TRANSBINDING TM NFkappaB ASSAY KIT)
RPN800 TNF IL-1 HIV mock medium RPN 756 (0.5, 1pgP24/cell) FIGURE 3BDETECTION OF PYK-2 PROTEIN TYROSINE KINASE IN CELL EXTRACT (BY W-B AFTER 30 MIN OF HIV EXPOSURE OF HFA)ASTROCYTES INFECTED WITH ADENOVIRUS MUTANT MT32IkB (2HR PRIOR HIV EXPOSURE) FIGURE 5 A, B, CCell-mediumCell-MockCell-IL1Cell-HIVAd.vec-mediumAd.vec-IL1Ad.vec-HIVAd.vec-MockAd.IkBa-mt32-mediumAd.IkBa-mt32 -MockAd.IkBa-mt32 -HIVAd.IkBa-mt32 -IL1IMMUNOSTAINING FOR DETECTION OF p65 SUBUNITS ACTIVATED NFkappaB(images from Confocal Laser Scanning Microscope LSM multiphoton 510)
LUCLUCLUCLUCLUCLUCLUCXXXXXFIGURE 4A THE DELETION ANALYSIS OF C3 PROMOTER REGION IN HIV RESPONSE OF PRIMARY ASTROCYTES FIGURE 4BTHE MUTATION ANALYSIS OF C3 PROMOTER IN HIV RESPONSE 1. mutation C1 109 -(-90) IL-1/IL-6 2. mutation C2 106- (-100) IL6 3. mutation C3 116 (-100) IFN- gamma 4. mutation C4 181- (-175) IL6 5. mutation C5 615 (-604) NFkB 6. mutation C6 750 (-740) NFkBCONCLUSIONSEXPOSURE OF PRIMARY ASTROCYTES TO HIV
INDUCES ACTIVATION of COMPLEMENT C3 PROMOTER, INCREASED C3 mRNA TRANSCRIPTION, and SECRETION of C3 PROTEIN
INDUCTION of C3 GENE requires primarily PROLINE RICH PROTEIN TYROSINE KINASE PYK-2 , MAPK signaling pathways, and is NFkappaB dependent; through indirect mechanism- NFkB binding sites in C3 promoter are dispensable for promoter activity
CRITICAL RESPONSIVE ELEMENT on C3 PROMOTER in HIV-1 RESPONSE is LOCATED; between NUCLEOTIDES [-109- (90)] with SUBDOMAIN at [ 106- (-100]; and it includes IL1-, and IL-6 responsive elementsXINDUCTION OF C3 PROMOTER TESTED IN CELL EXTRACTDETECTION OF p50 SUBUNITS OF ACTIVATED NFkappaB HETERODIMER IN NUCLEAR EXTRACT (30MIN/HIV)FIG 1AFIG 1BFIG 1CFIG 2AFIG 2BFIG 2CFIG 5AFIG 5BFIG 5C
Chart1
1.5333333330.251661148
1.8750.275
3.190.69
0.8750.075
1.010.01
48h
C3 Promoter Activity (RLU, FC)
inhibitors 2b graph
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graph cont inhib 2b
1577.9917269723
62.52
113.6055512755
150.2385979883
48h
cell extract
Relative Luciferase Activity [RLU]
kontrole do inhib 2b
HIVmedmock cIL-1TNF-mediummock c
average31111515762.51115
25.51316156.5551316
40.011118158.170.531118
26.95911156.461.97911
average30.82111515762.51115
SD7.991726972323.60555127550.95393920147.778553850223.6055512755
Ttest0.03534436910.00000031680.0006591335
IL-1 betaTNF-alfamediummock
15762.51115
156.5551316
158.170.531118
156.461.97911
av15762.51115
SD0.95393920147.778553850223.6055512755
0.0006591335
inhibitors2b
HIVIL-1 betaTNF- alfamediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium
3115762.5111533.61311.51111.43733382137.6302825
7.99172697230.95393920147.778553850223.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111
19-Jul
HIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumCalphostin/HIVCalphostin/medium
av31111533.61311.51111.437332825
SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031356.55743852432.6457513111
controle do graph inhib2b wl
controle do graph inhib2b wl
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
wl graph 1 Elisa p50NFkB
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graphknock 5000vpC3 prom
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
wlgraph knockout Il1 Il6 5000vp
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
knockout Il1, Il6 5000vp
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
knockout5000Il1,Il6
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
wl graph ad 5000 C3 prom bz Il1
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
daneknockout 5000vp promotor C3
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
1 EL p50NFkB
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
EL p50 controls
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
mC3RNA 24h, 3d signal transd
mC3RNA 24h, 3d signal transd
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
inhib2A wlasc grapg bez Il1
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
graph Inhib 2A bez Il1
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
inhibitors 2A
inhibitors 2A
1312
451.5275252317
222.5166114784
20.61.443375673
9.90.1
5.30.1527525232
6.20.2645751311
4.180.2306512519
124.31.5275252317
461
6.80.3511884584
6.30.2
48h
cell extract
Relative Luciferase Activity [RLU]
grph C3 protein1
1312
451.5275252317
222.5166114784
20.61.443375673
9.90.1
5.30.1527525232
6.20.2645751311
4.180.2306512519
124.31.5275252317
461
6.80.3511884584
6.30.2
48h
cell extract
Relative Luciferase Activity [RLU]
daneC3 proteina
1312
451.5275252317
222.5166114784
20.61.443375673
9.90.1
5.30.1527525232
6.20.2645751311
4.180.2306512519
124.31.5275252317
461
6.80.3511884584
6.30.2
48h
cell extract
Relative Luciferase Activity [RLU]
Q-PCR 24, 7ddane
Exp 02/05IL-1 beta 1ng/ml][20ng/ml]Ad-IkB/IL-1Ad-IkB/IL-6Ad-IkB/mockAdenovirusAd/mockAd/IL-1
Br52/2
systemIL-1 betaIL-6mockmediumAd-IkB/IL-1Ad-IkB/IL-6Ad-IkB/mockAdenovirus-IkB/mediumAd/IL-1Ad/IL-6Ad/mockAdenovirus/medium
average131452220.69.95.36.24.18124.3466.86.3
11314422219.75.56.63.94125457.26.5
2129471918.59.55.36.14.4126476.86.3
31334624219.65.26.54.2123466.56.1
average13145.666666666722.2520.6259.65.33333333336.44.18124.6666666667466.83333333336.3
SD21.52752523172.51661147841.4433756730.10.15275252320.26457513110.23065125191.527525231710.35118845840.2
Ttest
Ad-IkB/mockAdenovirus-IkBAdenovirusAd/mockAd/IL-1Ad/IL-6
6.97.1124.346
Ad-IkB/mockAdenovirus-IkB/medium6.57.212545
6.24.256.36.812647
6.63.946.16.512346
6.14.46.36.8333333333124.666666666746
6.54.20.20.35118845841.52752523171
6.44.18
0.26457513110.2306512519
Q-PCR 24, 7ddane
1262
451.5275252317
242.5166114784
221.443375673
9.90.1
5.70.1527525232
4.250.2306512519
6.20.2645751311
6.90.2
7.10.3511884584
124.31.5275252317
461
5000vp/cell
cell extract
Relative Luciferase Activity [RLU]
wykresy Q-PCR mRNA24, 7d
wykresy Q-PCR mRNA24, 7d
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
wl wykr C3 promoter
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
wykr promoter C3 ind
Exp 02/05Ad 5000vp/cellAd-IkB/mockAd-IkB/HIV
Br52/2Adenovirus-IkB
systemHIV+mockmediumAdenovirus-IkBAd-IkB/mockAd-IkB/HIVAdenovirusAd/mockAd/HIV+
average4724224.256.24.96.97.138.3
14822213.946.64.76.57.236
2491918.54.46.14.96.36.837
34624214.26.54.66.16.539
average47.522.2520.6254.186.44.73333333336.36.833333333337.3333333333
SD1.52752523172.51661147841.4433756730.23065125190.26457513110.15275252320.20.35118845841.5275252317
Br52/2
systemHIV+mockmediumAd-IkB/HIV+Ad-IkB/mockAd-IkB/mediumAd/HIV+Ad/mockAd-medium
average4724224.96.24.2538.37.16.9
14822214.76.63.94367.26.5
2491918.54.96.14.4376.86.3
34624214.66.54.2396.56.1
average47.522.2520.6254.73333333336.44.1837.33333333336.83333333336.3
SD1.52752523172.51661147841.4433756730.15275252320.26457513110.23065125191.52752523170.35118845840.2
dane i wykr promoter C3
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
Elisa NFkB controls z knockout
systemcontrol+Competition cIL-1 betaIL-6HIV+mockmedium
average0.2170.0330.2040.1240.1560.0620.047
10.2170.2170.1240.160.060.047
20.2140.0360.2080.1210.150.0610.044
30.220.0330.2040.1250.1620.0640.049
average0.2170.03450.20966666670.12333333330.15733333330.06166666670.0466666667
SD0.0030.00212132030.00665832810.0020816660.00642910050.0020816660.0025166115
HIV [2pg]HIV [4pg]TNF[10U/ml]IL-1[5ng/ml]control extmediumpositive controlcompetition control
av0.20.30.350.530.0270.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333
SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503
Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007
HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control
av0.280.370.40.530.0260.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.28666666670.37333333330.39666666670.52666666670.02566666670.02366666670.5423333333-0.0103333333
SD0.01154700540.02516611480.00577350270.00577350270.00057735030.00057735030.00251661150.0005773503
Ttest0.00000255570.00001811070.00000000.00000000.00000000.0000002
wykres Elisa NFkB contr z Knock
cell signalling involved in the activation of C3 gene expression
HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium
24h1410.672.90.580.4931.170.96.30.630.69
13.70.90.592.40.550.512.61.280.85.80.90.77
15.21.10.712.20.610.522.91.41.15.70.860.82
av14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76
SD0.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852
test
3d15.540.6560.783.50.40.93.12.11.86.70.780.5
16.40.70.793.40.520.913.22.21.66.40.980.6
16.30.630.822.950.490.893.52.31.56.20.890.67
av16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59
SD0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375
SD
HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium
24h14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76
3d16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59
SD240.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852
SD3d0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375
wykres Elisa NFkB contr z Knock
14.316.08350.79372539330.47031904071
10.6620.93333333330.10.0353836120.1527525232
0.65666666670.79666666670.96333333330.06110100930.020816660.118462371
2.53.28333333333.33333333330.36055512750.29297326390.4509249753
0.580.470.75666666670.030.062449980.0585946528
0.50666666670.90.80.01527525230.010.1
2.83333333333.26666666674.10.20816659990.20816659990.1732050808
1.28333333332.22.46666666670.11503622620.10.2516611478
0.93333333331.63333333332.09333333330.15275252320.15275252320.1101514109
5.93333333336.433333333311.40.32145502540.25166114781.1532562595
0.79666666670.88333333330.91333333330.145716620.1001665282.74
0.760.590.13233333330.06557438520.08544003750.0924463809
24h
3d
7d
cell extract
Relative quantification; Fold change
Cell signalling involved in the activation of C3 gene expression
OD dane NFkB
000.79372539330.4703190407
000.10.035383612
000.06110100930.02081666
000.36055512750.2929732639
000.030.06244998
000.01527525230.01
000.20816659990.2081665999
000.11503622620.1
000.15275252320.1527525232
000.32145502540.2516611478
000.145716620.100166528
000.06557438520.0854400375
24h
3d
cell extract
Fold change [C3 mRNA]
neutralization of Il6
neutralization of Il6
135.33333333336.1101009266
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Re;ative Luciferase Activity [RLU]
Inhib December grap1 July 20,07
135.33333333336.1101009266
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Re;ative Luciferase Activity [RLU]
GrapInhDec July 20,07
HIVmockmediumCape/HIVCape/mockCape/mediumP38/HIVP38/mockP38/mediumSB580/HIVSB580/mockSB580/mediumSB190/HIVSB190/mockSB580/mediumAG17/HIVAG17/mockAG17/mediumJNK/HIVJNK/mockJNK/mediumGenistein/HIVGenistein/mockGenistein/medium
135.33333333336566.33333333335.56666666675.55.5333333333100.333333333311185.333333333333.333333333337.666666666733.33333333332121.666666666721.666666666753.666666666751.66666666674965.666666666747.3333333333493.66666666675.33333333333.6666666667Average
6.11010092664.35889894351.52752523170.51316014390.50.923760430717.6162803498.18535277198.96288643984.93288286238.504900548110.40832999734.35889894358.96288643988.08290376875.50757054736.65832811855.291502622111.01514109462.51661147842.64575131110.57735026920.57735026920.5773502692SD
0.00008431090.00001958870.00001947480.00002075310.02798383280.00100840240.024151360.00113383070.00774020590.00827556490.00024601170.00122031550.04907193280.01557401090.00690512192.51661147840.0055620.00001740970.00001942550.0000174097Ttest
GrapInhDec July 20,07
135.33333333336.1101009266
37916.8226038413
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Relative Luciferase Activity [RLU]
Inhibitors December or1 graph
1NFkB ELISA
PROTEINAVERAGESD
IL-1141.66666677.637626158
TNF-alfa108.66666673.214550254
HIV [0.5]10.333333331.040833
HIV [1pg]19.166666672.020725942
HIV [2.5pg]20.766666671.078579312
mock0.30.2
medium0.2433333330.049328829
IL-1512.66666676.429100507
TNF-alfa457.76666678.494900431
HIV [0.5]87.333333333.214550254
HIV [1pg]191.66333336.345079458
HIV [2.5pg]236.666666712.58305739
mock3.510.5
medium30.1
IL-1 betaTNF-alfaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium
av3d141.66108.6610.3319.1620.760.30.25
av7d512.66457.7687.33191.66236.661412.5
SD7.63.21.0421.0732
Sd6.48.43.26.312.532.5
IL-1HIV [0.5pg]HIV [1pg]HIV [2.5 pg]mock cmediumIL-1HIV [0.5]HIV [1pg]HIV [2.5 pg]mock cmedium
average10.33333333333.24.85.33333333331.066666666718.76666666673.53333333334.26666666673.73333333331.23333333331.1666666667
SD0.57735026920.11.31148770490.28867513460.20816659990.36055512750.49328828620.35118845840.30550504630.46188021540.11547005380.1154700538
Ttest0.0000127030.00008921650.00822227110.00003178290.00001350040.0004205250.00008733320.0008104185
IL-1 betaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium
av24h13.333.24.85.5511.1
av7d93.64.36.51.21.6
SD0.570.11.30.20.20.4
SD0.50.350.30.40.20.1
HIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium
24h3.24.85.5511.1
7d3.64.36.51.21.6
SD0.11.30.20.20.4
SD0.350.30.40.20.1
10.33333333330.5773502692
3.20.1
4.81.3114877049
5.33333333330.2886751346
1.06666666670.2081665999
10.3605551275
8.76666666670.4932882862
3.53333333330.3511884584
4.26666666670.3055050463
3.73333333330.4618802154
1.23333333330.1154700538
1.16666666670.1154700538
48h
cell extract
Fold change [Q-PCR]
000.570.5
000.10.35
001.30.3
000.20.4
000.20.2
000.40.1
24h
7d
cell extract
C3 mRNA (FC)
000.10.35
001.30.3
000.20.4
000.20.2
000.40.1
24h
7d
mRNA (Fold Change)
13.3390.570.5
3.23.60.10.35
4.84.31.30.3
5.556.50.20.4
11.20.20.2
1.11.60.40.1
24h
7d
cell extract
Fold hange [mRNA]
13.3390.570.5
3.23.60.10.35
4.84.31.30.3
5.556.50.20.4
11.20.20.2
1.11.60.40.1
24h
7d
cell extract
Fold hange [mRNA]
9.750.75
6.30.7
1.5333333330.251661148
1.8750.275
3.190.69
0.8750.075
1.010.01
48h
cell extract
Fold change [RLU]
9.750.75
6.30.7
1.5333333330.251661148
1.8750.275
3.190.69
0.8750.075
1.010.01
48h
cell extract
Fold change [RLU]
9.750.75
6.30.7
1.5333333330.251661148
1.8750.275
3.190.69
0.8750.075
1.010.01
48h
cell extract
Fold change [RLU]
9.750.75
6.30.7
1.5333333330.251661148
1.8750.275
3.190.69
0.8750.075
1.010.01
48h
cell extract
Fold change [RLU]
IL-1betaTNF-alfaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium
9.756.31.5333333331.8753.190.8751.01
SD0.750.70.2516611480.2750.690.0750.01
00.251661148
00.275
00.69
00.075
00.01
48h
Relative Luciferase Activity [RLU] Fold Change
0.2170.003
0.0330.0021213203
0.2040.0066583281
0.1560.0064291005
0.0620.002081666
0.0470.0025166115
0.0560.0026457513
0.060.001
0.040.01
nuclear extract
OD value [450nm]
0.2170.003
0.0330.0021213203
0.2040.0066583281
0.1560.0064291005
0.0620.002081666
0.0470.0025166115
0.0560.0026457513
0.060.001
0.040.01
nuclear extract
OD value [450nm]
0.2170.003
0.0330.0021213203
0.2040.0066583281
0.1560.0064291005
0.0620.002081666
0.0470.0025166115
0.0560.0026457513
0.060.001
0.040.01
nuclear extract
OD value [450nm]
controls
osobno IL-1, IL-6
systemcontrol+Competition cIL-1 betaHIV+mockmediumCape/HIV+Cape/mockCape/medium
average0.2170.0330.2040.1560.0620.0470.0560.060.04
10.2170.2170.160.060.0470.0540.060.03
20.2140.0360.2080.150.0610.0440.0550.0590.05
30.220.0330.2040.1620.0640.0490.0590.0610.04
average0.2170.03450.20966666670.15733333330.06166666670.04666666670.0560.060.04
SD0.0030.00212132030.00665832810.00642910050.0020816660.00251661150.00264575130.0010.01
wirusy
systemcontrol+Competition cmockmediumHIV+Ad-v/HIV+Ad-v/mockAd-vAd-IkB/HIV+Ad-IkB/mockAd-IkBCape/HIVCape/mock
average0.2170.0330.0620.0470.1560.1320.1130.1010.0510.0530.0490.0560.06
10.2170.060.0470.160.1340.1150.1080.0560.0530.050.0540.06
20.2140.0360.0610.0440.150.1380.1180.0990.0530.0560.0470.0550.059
30.220.0330.0640.0490.1620.1240.1060.0960.0440.050.050.0590.061
average0.2170.03450.06166666670.04666666670.15733333330.1320.1130.1010.0510.0530.0490.0560.06
SD0.0030.00212132030.0020816660.00251661150.00642910050.00721110260.0062449980.0062449980.0062449980.0030.00173205080.00264575130.001
0.2170.003
0.0330.0149899967
0.2040.0066583281
0.1240.002081666
0.1560.0357258077
0.1320.0072111026
0.1130.006244998
0.1010.006244998
0.0510.006244998
0.0530.003
0.0490.0017320508
0.0560.0026457513
0.060.001
0.0620.002081666
0.0470.0025166115
cell extract
OD value [450nm]
IL-6HIVmockmediummouse IgGMoAb anti-IL6mouse IgG+HIVMoAb anti-IL6+HIV
Fch1.81.510.91.41.041.71
SD
4.52.82.71.83.92.3
42.72.82.23.62.1
3.73.22.662.23.31.8
SD0.40.20.20.150.30.260.40.28
Fold Ch1.81.510.91.41.041.71
1.80.4
1.50.2
10.2
0.90.15
1.40.3
1.040.26
1.70.4
10.28
48h
cell extract
Fold change [RLU]
1.80.4
1.50.2
10.2
0.90.15
1.40.3
1.040.26
1.70.4
10.28
48h
cell extract
Fold change [RLU]
1570.9539392014
62.57.7785538502
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.7
11.50.5
110.8857388629
11.42.1166010489
430.7110365087
300.4150803135
385.3605596723
210.8669486721
37.64.8967233943
304.4303047299
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
1570.9539392014
62.57.7785538502
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.7
11.50.5
110.8857388629
11.42.1166010489
430.7110365087
300.4150803135
385.3605596723
210.8669486721
37.64.8967233943
304.4303047299
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
IL-1TNF-HIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium
15762.5average31111533.61311.51111.43733382137.6302825
156.55525.513163.1234.01213.091212.1936323320.4632.7834.462724
158.170.5340.0111182.7253.38413.0911.510.712.2353343.662242.5725.62223
156.461.9726.959113.1523.40412.811110.461336.732.637.3420.5437.4529.943528
15762.5average30.82111533.612.996666666711.511.086666666711.44330382137.6302825
0.95393920147.7785538502SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111
0.00000320.0006591335Ttest0.03534436910.00452958730.00550742530.01506078120.02077571580.01074868790.21012594370.09889791720.83434125080.0035112510.04869066430.0029959170.01043022450.03959261940.0179479132
tosamo z Il1
IL-1 betaTNF- alfaHIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium
15762.531111533.61311.51111.44330382137.6302825
156.55525.513163.1234.01213.091212.1936323320.4632.7834.462724
158.170.5340.0111182.7253.38413.0911.510.712.2353343.662242.5725.62223
156.461.9726.959113.1523.40412.811110.461336.732.637.3420.5437.4529.943528
15762.530.82111533.612.996666666711.511.086666666711.44330382137.6302825
SD0.95393920147.77855385027.991726972323.60555127550.23859798830.35694257240.70.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111
0.00000320.00065913350.03534436910.00452958730.00550742530.01506078120.02077571580.01074868790.21012594370.09889791720.83434125080.0035112510.04869066430.0029959170.01043022450.03959261940.0179479132
HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control
0.20.30.350.530.0270.0240.545-0.01
0.220.320.360.550.0230.0220.546-0.011
0.210.330.3670.60.0210.0230.546-0.012
av
SD
MBD0002EB81.xls
C3protein wykres or
141.66512.667.66.4
108.66457.73.28.4
10.3387.331.043.2
19.16191.6626.3
20.762371.0712.5
0.311.533
0.2410.5
C
3d
7d
supernatants
C3 Protein [ng/ml]
inhibitors 2b graph
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graph cont inhib 2b
1577.9917269723
62.52
113.6055512755
150.2385979883
48h
cell extract
Relative Luciferase Activity [RLU]
kontrole do inhib 2b
HIVmedmock cIL-1TNF-mediummock c
average31111515762.51115
25.51316156.5551316
40.011118158.170.531118
26.95911156.461.97911
average30.82111515762.51115
SD7.991726972323.60555127550.95393920147.778553850223.6055512755
Ttest0.03534436910.00000031680.0006591335
IL-1 betaTNF-alfamediummock
15762.51115
156.5551316
158.170.531118
156.461.97911
av15762.51115
SD0.95393920147.778553850223.6055512755
0.0006591335
inhibitors2b
HIVIL-1 betaTNF- alfamediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium
3115762.5111533.61311.51111.43733382137.6302825
7.99172697230.95393920147.778553850223.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111
19-Jul
HIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumCalphostin/HIVCalphostin/medium
av31111533.61311.51111.437332825
SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031356.55743852432.6457513111
controle do graph inhib2b wl
controle do graph inhib2b wl
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
wl graph 1 Elisa p50NFkB
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graphknock 5000vpC3 prom
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
wl graph ad 5000 C3 prom bz Il1
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
daneknockout 5000vp promotor C3
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
1 EL p50NFkB
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
EL p50 controls
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
mC3RNA 24h, 3d signal transd
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
inhib2A wlasc grapg bez Il1
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graph Inhib 2A bez Il1
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
C3 protein wykres
C3 protein wykres
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
inhibitors 2A
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
C3 proteimna graph or
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
C3 proteina
C3 proteina
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
1NFkB ELISA
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
Exp 02/05Ad 5000vp/cellAd-IkB/mockAd-IkB/HIV
Br52/2Adenovirus-IkB
systemHIV+mockmediumAdenovirus-IkBAd-IkB/mockAd-IkB/HIVAdenovirusAd/mockAd/HIV+
average4724224.256.24.96.97.138.3
14822213.946.64.76.57.236
2491918.54.46.14.96.36.837
34624214.26.54.66.16.539
average47.522.2520.6254.186.44.73333333336.36.833333333337.3333333333
SD1.52752523172.51661147841.4433756730.23065125190.26457513110.15275252320.20.35118845841.5275252317
Br52/2
systemHIV+mockmediumAd-IkB/HIV+Ad-IkB/mockAd-IkB/mediumAd/HIV+Ad/mockAd-medium
average4724224.96.24.2538.37.16.9
14822214.76.63.94367.26.5
2491918.54.96.14.4376.86.3
34624214.66.54.2396.56.1
average47.522.2520.6254.73333333336.44.1837.33333333336.83333333336.3
SD1.52752523172.51661147841.4433756730.15275252320.26457513110.23065125191.52752523170.35118845840.2
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
systemcontrol+Competition cIL-1 betaIL-6HIV+mockmedium
average0.2170.0330.2040.1240.1560.0620.047
10.2170.2170.1240.160.060.047
20.2140.0360.2080.1210.150.0610.044
30.220.0330.2040.1250.1620.0640.049
average0.2170.03450.20966666670.12333333330.15733333330.06166666670.0466666667
SD0.0030.00212132030.00665832810.0020816660.00642910050.0020816660.0025166115
HIV [2pg]HIV [4pg]TNF[10U/ml]IL-1[5ng/ml]control extmediumpositive controlcompetition control
av0.20.30.350.530.0270.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333
SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503
Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007
HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control
av0.280.370.40.530.0260.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.28666666670.37333333330.39666666670.52666666670.02566666670.02366666670.5423333333-0.0103333333
SD0.01154700540.02516611480.00577350270.00577350270.00057735030.00057735030.00251661150.0005773503
Ttest0.00000255570.00001811070.00000000.00000000.00000000.0000002
cell signalling involved in the activation of C3 gene expression
HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium
24h1410.672.90.580.4931.170.96.30.630.69
13.70.90.592.40.550.512.61.280.85.80.90.77
15.21.10.712.20.610.522.91.41.15.70.860.82
av14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76
SD0.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852
test
3d15.540.6560.783.50.40.93.12.11.86.70.780.5
16.40.70.793.40.520.913.22.21.66.40.980.6
16.30.630.822.950.490.893.52.31.56.20.890.67
av16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59
SD0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375
SD
HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium
24h14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76
3d16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59
SD240.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852
SD3d0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375
14.316.08350.79372539330.47031904071
10.6620.93333333330.10.0353836120.1527525232
0.65666666670.79666666670.96333333330.06110100930.020816660.118462371
2.53.28333333333.33333333330.36055512750.29297326390.4509249753
0.580.470.75666666670.030.062449980.0585946528
0.50666666670.90.80.01527525230.010.1
2.83333333333.26666666674.10.20816659990.20816659990.1732050808
1.28333333332.22.46666666670.11503622620.10.2516611478
0.93333333331.63333333332.09333333330.15275252320.15275252320.1101514109
5.93333333336.433333333311.40.32145502540.25166114781.1532562595
0.79666666670.88333333330.91333333330.145716620.1001665282.74
0.760.590.13233333330.06557438520.08544003750.0924463809
24h
3d
7d
cell extract
Relative quantification; Fold change
Cell signalling involved in the activation of C3 gene expression
0.79372539330.4703190407
0.10.035383612
0.06110100930.02081666
0.36055512750.2929732639
0.030.06244998
0.01527525230.01
0.20816659990.2081665999
0.11503622620.1
0.15275252320.1527525232
0.32145502540.2516611478
0.145716620.100166528
0.06557438520.0854400375
24h
3d
cell extract
Fold change [C3 mRNA]
135.33333333336.1101009266
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Re;ative Luciferase Activity [RLU]
135.33333333336.1101009266
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Re;ative Luciferase Activity [RLU]
141.66512.66
108.66457.7
10.3387.33
19.16191.66
20.76237
0.311.5
0.2410.5
3d
7d
supernatants
C3 Protein [ng/ml]
141.66512.66
108.66457.7
10.3387.33
19.16191.66
20.76237
0.311.5
0.2410.5
3d
7d
supernatants
C3 Protein [ng/ml]
HIVmockmediumCape/HIVCape/mockCape/mediumP38/HIVP38/mockP38/mediumSB580/HIVSB580/mockSB580/mediumSB190/HIVSB190/mockSB580/mediumAG17/HIVAG17/mockAG17/mediumJNK/HIVJNK/mockJNK/mediumGenistein/HIVGenistein/mockGenistein/medium
135.33333333336566.33333333335.56666666675.55.5333333333100.333333333311185.333333333333.333333333337.666666666733.33333333332121.666666666721.666666666753.666666666751.66666666674965.666666666747.3333333333493.66666666675.33333333333.6666666667Average
6.11010092664.35889894351.52752523170.51316014390.50.923760430717.6162803498.18535277198.96288643984.93288286238.504900548110.40832999734.35889894358.96288643988.08290376875.50757054736.65832811855.291502622111.01514109462.51661147842.64575131110.57735026920.57735026920.5773502692SD
0.00008431090.00001958870.00001947480.00002075310.02798383280.00100840240.024151360.00113383070.00774020590.00827556490.00024601170.00122031550.04907193280.01557401090.00690512192.51661147840.0055620.00001740970.00001942550.0000174097Ttest
135.33333333336.1101009266
37916.8226038413
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Relative Luciferase Activity [RLU]
141.66512.66
108.66457.7
10.3387.33
19.16191.66
20.76237
0.311.5
0.2410.5
3d
7d
supernatants
C3 Protein [ng/ml]
PROTEINAVERAGESD
IL-1141.66666677.637626158
TNF-alfa108.66666673.214550254
HIV [0.5]10.333333331.040833
HIV [1pg]19.166666672.020725942
HIV [2.5pg]20.766666671.078579312
mock0.30.2
medium0.2433333330.049328829
IL-1512.66666676.429100507
TNF-alfa457.76666678.494900431
HIV [0.5]87.333333333.214550254
HIV [1pg]191.66333336.345079458
HIV [2.5pg]236.666666712.58305739
mock3.510.5
medium30.1
IL-1 betaTNF-alfaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium
AV3d141.66108.6610.3319.1620.760.30.24
Av7d512.66457.787.33191.6623711.510.5
SD3d7.633.21.042.021.070.20.04
SD7d6.428.43.26.312.53.52
HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control
0.20.30.350.530.0270.0240.545-0.01
0.220.320.360.550.0230.0220.546-0.011
0.210.330.3670.60.0210.0230.546-0.012
av
SD
MBD0009EEC5.xls
22,3
53
24
50
5.65
2.5
1.4
0.5
25
22
22.5
1-control(+)HIV, 2-Control(-)HIV, 3-Calphostin-C, 4-SQ22536, 5-CAPE 1:10000, 6-CAPE 1:15000, 7-Genistein 1:1000, 8-Genistein 1:2000, 9-PMA, 10-Pertusis toxin, 11-8Br-cAMP
inhibitors
RATIO pGLC3pro/pRLTK dual luciferase assay
Br22/3 C3 PROMOTER INDUCTION by HIV with INHIBITORS
22,3+-
5312
2421
5033
5.654.5
2.54.6
1.40.98
0.50.4
2522
2215
22.515
contr(+)/(-)
Calph
SQ22536
CAPE:10000
CAPE;15000
Genist:1000
Genist:2000
PMA
Pertusis toxin
8Br-cAMP
+HIV
-HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK Dual Luciferase Assay
BRAIN 22/3
Sheet1
5312
2421
5033
5.654.5
2.54.6
1.40.98
0.50.4
2522
2215
22.515
Sheet1
0
0
0
0
0
0
0
0
0
0
0
inhibitors(-)HIV
inhibitors
Ratio pGlC3/pRLTH
C3 induction promoter - inhibitors without HIV
Br22,4
Br22,4
49
25
16.5
44
18
7.6
4.7
1.2
20
22
31.4
35
Br22/4 inhibitors (+) HIV
inhibitors
Ratio pGL3C/pRLTK
-
20
22.5
30
33.5
11
9.7
1.3
3.4
20
20
32
25
inhibitors(+) HIV
inhibitors
Ratio pGLC3/pRLTK Dual-Luciferase Assay
BR 22/4 C3 induction promoter
49
25
16.5
44
18
7.6
4.7
1.2
20
22
31.4
35
inhibitors (-) HIV
18,4
50
46
26
42
18.4
126
84
72.5
75.6
inhibitors (+) HIV
inhibitors
Ratio pGLC3/pRLTK
Br 18/5 C3 induction promoter
18,4 bez HIV
23
42
35
56
16
72
45
80
38
inhibitors (-) HIV
br4p4
4920
2522.5
16.530
4433.5
1811
7.69.7
4.71.3
1.23.4
2020
2220
31.432
3525
control(+)/(-)
Calph:1000
Calph10-5
SQ22536
CAPE:20000
CAPE:15000
Genist:2000
Genist:1000
8-Br-cAMP(1pgHIV)
PMA(1pgHIV)
Pertusis t1x
Pertusis t5x
(+)HIV
(-)HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK
Brain 22/4
C 3 promoter induction March06
7.5
4.5
2.5
3.8
1
cell extract
Fold change [Relative Luciferase Unit
C3 promoter induction by HIV in HFA
MVP and C3promoter
7.5
4.5
2.5
3.8
1
cell extract
Fold change [Luciferase Activity Unit]
C3 promoter induction by HIV[MVP] in HFA
Sheet14
IL1-beta[10ng/ml]TNF[10U/ml]MVP[2pgP24/cell]MVP 5pgP24/cellcontrol ext.
7.54.52.53.81
18,4por
5023
4642
2635
4256
18.416
12672
8445
72.580
75.638
control(+)/(-)
Calph
CAPE :20000
CAPE :50000
Genist:2000
8-Br-c-AMP(0.5 pg ADA)
PMA
SQ22536
Pertusis toxin
(+)HIV
(-)HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK
BRAIN 18/5
oct11
4421
3515
7737
1812
6045.6
3833
3016.6
1816
4641
1.61
2.51.6
2515
+HIV/HIV-
+HIV
Daid.
Wort.
PDTC
U126
SB202190
SB203580
SP
PT
CAPE
Gen
Calp
inhibitors with/w-oHIV
Ratio pGLC3pro/RLTK
Brain22/4 inhib part1and2 October11
br22part2inh
4421
3515
7737
1812
6045.6
3833
3016.6
1816
4641
1.61
2.51.6
2515
Sheet2
492050237012
2522.546427030
16.5302635112171
4433.542562.42
181118.41645
7.69.7126728488
4.71.3844514090
1.23.472.580113150
202075.638147130
22202643
31.4325158
35254072
82107
107262
159130
45104
6451
Sheet2
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
+HIV
-HIV
inhibitors with/or without HIV
RATIO pGLC3/RLTK
BRAIN 22/3 # NOV12
Il1 beta Jims
160
126
125
Jims stock U/ml
Influence of IL1-beta on induction C3 promoter
C 3 gene expr 48h
8.68
12.3
1
0.6
48h
cell extract
Fold change
C3 gene expression in HFA by HIV
Mouse signal transduction study
217
130
140
38
60
47
227
206
100
11
14
11
17
15
20
59
80
78
340
cell extract
Fold change
Effect of signal transduction's inhibitors on C3 induction promoter by HIV in primary mouse astrocytes
C3 promoter and mouse 2
238
152
138
1.5
1.3
46
35
20
44
17.5
23
25
80
122
104
173
193
227
340
cell extract
Fold change
Effect of signal transduction's inhibitors on C3 promoter induction by HIV in primary mouse astrocytes
Sheet11
NDKcontrol extmediumSB 190/NDKSB190/control extSB190/mediumCalphostion/NDKCalphostin/control ext.Calphostin/mediumJNK inh/NDKJNKinh/cont.extJNK/mediumPDTC/NDKPDTC/control ext.PDTC/mediumSB580/NDKSB580/control extSB580/mediumIL/TNF
217130140386047227206100111411171520598078340
NDKcontrol extmediumCape/NDKCape/control extCape/mediumGenistein/NDKGenistein/control extGenistein/mediumPD 98/NDKPD98/control extPD 98/mediumAG18/NDKAG18/control extAG18/mediump38/NDKp38/control extp38/mediumIL/TNF
2381521381.51.34635204417.5232580122104173193227340
mouse and inhibitors
217.5
130
140
SB190
38
60
47
SB580
59
80
78
Calphostin
227
206
100
JNK
11
14
11
PDTC
17
15
20
SQ
130
170
144
Pertusis toxin
236
225
205
PD 98
17.5
23
25
AG18
80
122
104
P38
173
193
227
MFA
cell extract
Relative Luciferase Activity [RLA]
Effect of signal transduction inhibitors on C3 promoter induction by HIV in primary mouse astrocytes
Sheet10
NDK217.5
c.ext130
medium140
SB190
+ NDK38
c.ext60
medium47
SB580
+ NDK59
c.ext80
medium78
Calphostin
+ NDK227
c.ext206
medium100
JNK
+ NDK11
c.ext14
medium11
PDTC
+ NDK17
c.ext15
medium20
SQ
+ NDK130
c.ext170
medium144
Pertusis toxin
+ NDK236
c.ext225
medium205
PD 98
+ NDK17.5
c.ext23
medium25
AG18
+ NDK80
c.ext122
medium104
P38
+ NDK173
c.ext193
medium227
gene expr 7 days
37.6
3378.5
1
0.84
7 days
cell extract
Fold change
C 3 gene expression in HFA by HIV
Sheet9
MVP[5pgP24/cellIL1-beta[10ng/ml]control extractmedium
37.63378.510.84
MVP[5pgP24/cell]IL1-beta[10ng/ml]control extractmedium
8.6812.310.6
Sheet8
1.0430.1891.0150.545
C3 gene exp 7days
4.84
37.6
1
376.7
3378.6
7 days
cell extract
fold change
C3 gene expression by HIV {MVP} in HFA on 7 days of infection
Sheet7
7days
MVP [J]1pg5pgc.extTNFIL1
4.8437.61376.73378.6
Sheet4
12negative16020U/ml13720ng/ml138
10720012610U/ml11910ng/ml119
601001250.2U/ml1382ng/ml137
5650
Sheet4
0
0
0
0
-/+HIV
Ratio pGLC3pro/RLTK
Induction of C3 promoter by HIV dose dependent
C3 gene expr march24
0
0
0
Jim's stock
Il 1-beta [U/ml]
Ratio pGLC3 pro/RLTK
Influence of IL1-beta on C3 induction promoter
Sheet13
0
0
0
New Il1-beta
IL1-beta [ng/ml]
Ratio pGLC3pro/RLTK
Influence of Il1-beta on C3 induction promoter
p50 NFkB BR37
1.68
2.82
2.44
3.67
1
1.04
24h
Cell extract
Fold change
C3 gene expresion in HFA by HIV
p50-p65p
Panomic dec12
0
00.2
00.3
00.35
00.53
00.027
00.024
00.545
0-0.01
cell extract
OD [p50-p65]
Detection of p50 subunits of heterodimer p50-p65 NFkB in HFA after HIV infection
Sheet12
0.2
0.3
0.35
0.53
0.027
0.024
0.545
-0.01
OD [NFkBp50]
Sheet6
0.20.0529150262
0.30.05
0.350.0264575131
0.530.0057735027
0.0270.001
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
**
***
****
****
****
cell extract
OD [NFkB subunit p50]
Sheet5
HIV [2pg]HIV [4pg]TNF[10U/ml]IL-1[5ng/ml]control extmediumpositive controlcompetition control
av0.20.30.350.530.0270.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333
SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503
Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007
NDK
Sheet3
NDK fNDK nNL4-3 fIL/TNFc.ext 10ulmedium
1.682.822.443.6711.04
3.63.491
1.60.7614
5081
67120
6040
116.665
67.7
0
0
0
0
0
0
0
Cape exp
inhibitor/+-IL1beta/+-HIV
Ratio pGLC3pro/RLTK
CAPE/+-HIV/+-IL1beta
0
0
0
0
0
0
GENISTEIN exp
inhibitor/+-HIV/+_ IL1-beta
RATIO pGLC3pro/RLTK
GENISTEIN/+-HIV/+_IL1-beta
0
0
Controls- HFA brain28/3 infection NL4-3/PBL
HIV/-HIV
RATIO pGLC3pro/RLTK
149
2430
148.5184.4
119.25210.25
81153
52.2573.33
120206.66
14.6616.33
2741
96152.33
59.33125.33
71.6670
00
2438
00
00
00
00
00
00
00
Ratio
Effect of HIV on the activity of different C3 promoter constructs in HFA
00
00
00
00
00
00
00
Ratio
Effect of HIV on the activity of different C3 promoter constructs in HFA [systemA/2]
MBD0009EEC5.xls
22,3
53
24
50
5.65
2.5
1.4
0.5
25
22
22.5
1-control(+)HIV, 2-Control(-)HIV, 3-Calphostin-C, 4-SQ22536, 5-CAPE 1:10000, 6-CAPE 1:15000, 7-Genistein 1:1000, 8-Genistein 1:2000, 9-PMA, 10-Pertusis toxin, 11-8Br-cAMP
inhibitors
RATIO pGLC3pro/pRLTK dual luciferase assay
Br22/3 C3 PROMOTER INDUCTION by HIV with INHIBITORS
22,3+-
5312
2421
5033
5.654.5
2.54.6
1.40.98
0.50.4
2522
2215
22.515
contr(+)/(-)
Calph
SQ22536
CAPE:10000
CAPE;15000
Genist:1000
Genist:2000
PMA
Pertusis toxin
8Br-cAMP
+HIV
-HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK Dual Luciferase Assay
BRAIN 22/3
Sheet1
5312
2421
5033
5.654.5
2.54.6
1.40.98
0.50.4
2522
2215
22.515
Sheet1
0
0
0
0
0
0
0
0
0
0
0
inhibitors(-)HIV
inhibitors
Ratio pGlC3/pRLTH
C3 induction promoter - inhibitors without HIV
Br22,4
Br22,4
49
25
16.5
44
18
7.6
4.7
1.2
20
22
31.4
35
Br22/4 inhibitors (+) HIV
inhibitors
Ratio pGL3C/pRLTK
-
20
22.5
30
33.5
11
9.7
1.3
3.4
20
20
32
25
inhibitors(+) HIV
inhibitors
Ratio pGLC3/pRLTK Dual-Luciferase Assay
BR 22/4 C3 induction promoter
49
25
16.5
44
18
7.6
4.7
1.2
20
22
31.4
35
inhibitors (-) HIV
18,4
50
46
26
42
18.4
126
84
72.5
75.6
inhibitors (+) HIV
inhibitors
Ratio pGLC3/pRLTK
Br 18/5 C3 induction promoter
18,4 bez HIV
23
42
35
56
16
72
45
80
38
inhibitors (-) HIV
br4p4
4920
2522.5
16.530
4433.5
1811
7.69.7
4.71.3
1.23.4
2020
2220
31.432
3525
control(+)/(-)
Calph:1000
Calph10-5
SQ22536
CAPE:20000
CAPE:15000
Genist:2000
Genist:1000
8-Br-cAMP(1pgHIV)
PMA(1pgHIV)
Pertusis t1x
Pertusis t5x
(+)HIV
(-)HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK
Brain 22/4
C 3 promoter induction March06
7.5
4.5
2.5
3.8
1
cell extract
Fold change [Relative Luciferase Unit
C3 promoter induction by HIV in HFA
MVP and C3promoter
7.5
4.5
2.5
3.8
1
cell extract
Fold change [Luciferase Activity Unit]
C3 promoter induction by HIV[MVP] in HFA
Sheet14
IL1-beta[10ng/ml]TNF[10U/ml]MVP[2pgP24/cell]MVP 5pgP24/cellcontrol ext.
7.54.52.53.81
18,4por
5023
4642
2635
4256
18.416
12672
8445
72.580
75.638
control(+)/(-)
Calph
CAPE :20000
CAPE :50000
Genist:2000
8-Br-c-AMP(0.5 pg ADA)
PMA
SQ22536
Pertusis toxin
(+)HIV
(-)HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK
BRAIN 18/5
oct11
4421
3515
7737
1812
6045.6
3833
3016.6
1816
4641
1.61
2.51.6
2515
+HIV/HIV-
+HIV
Daid.
Wort.
PDTC
U126
SB202190
SB203580
SP
PT
CAPE
Gen
Calp
inhibitors with/w-oHIV
Ratio pGLC3pro/RLTK
Brain22/4 inhib part1and2 October11
br22part2inh
4421
3515
7737
1812
6045.6
3833
3016.6
1816
4641
1.61
2.51.6
2515
Sheet2
492050237012
2522.546427030
16.5302635112171
4433.542562.42
181118.41645
7.69.7126728488
4.71.3844514090
1.23.472.580113150
202075.638147130
22202643
31.4325158
35254072
82107
107262
159130
45104
6451
Sheet2
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
+HIV
-HIV
inhibitors with/or without HIV
RATIO pGLC3/RLTK
BRAIN 22/3 # NOV12
Il1 beta Jims
160
126
125
Jims stock U/ml
Influence of IL1-beta on induction C3 promoter
C 3 gene expr 48h
8.68
12.3
1
0.6
48h
cell extract
Fold change
C3 gene expression in HFA by HIV
Mouse signal transduction study
217
130
140
38
60
47
227
206
100
11
14
11
17
15
20
59
80
78
340
cell extract
Fold change
Effect of signal transduction's inhibitors on C3 induction promoter by HIV in primary mouse astrocytes
C3 promoter and mouse 2
238
152
138
1.5
1.3
46
35
20
44
17.5
23
25
80
122
104
173
193
227
340
cell extract
Fold change
Effect of signal transduction's inhibitors on C3 promoter induction by HIV in primary mouse astrocytes
Sheet11
NDKcontrol extmediumSB 190/NDKSB190/control extSB190/mediumCalphostion/NDKCalphostin/control ext.Calphostin/mediumJNK inh/NDKJNKinh/cont.extJNK/mediumPDTC/NDKPDTC/control ext.PDTC/mediumSB580/NDKSB580/control extSB580/mediumIL/TNF
217130140386047227206100111411171520598078340
NDKcontrol extmediumCape/NDKCape/control extCape/mediumGenistein/NDKGenistein/control extGenistein/mediumPD 98/NDKPD98/control extPD 98/mediumAG18/NDKAG18/control extAG18/mediump38/NDKp38/control extp38/mediumIL/TNF
2381521381.51.34635204417.5232580122104173193227340
mouse and inhibitors
217.5
130
140
SB190
38
60
47
SB580
59
80
78
Calphostin
227
206
100
JNK
11
14
11
PDTC
17
15
20
SQ
130
170
144
Pertusis toxin
236
225
205
PD 98
17.5
23
25
AG18
80
122
104
P38
173
193
227
MFA
cell extract
Relative Luciferase Activity [RLA]
Effect of signal transduction inhibitors on C3 promoter induction by HIV in primary mouse astrocytes
Sheet10
NDK217.5
c.ext130
medium140
SB190
+ NDK38
c.ext60
medium47
SB580
+ NDK59
c.ext80
medium78
Calphostin
+ NDK227
c.ext206
medium100
JNK
+ NDK11
c.ext14
medium11
PDTC
+ NDK17
c.ext15
medium20
SQ
+ NDK130
c.ext170
medium144
Pertusis toxin
+ NDK236
c.ext225
medium205
PD 98
+ NDK17.5
c.ext23
medium25
AG18
+ NDK80
c.ext122
medium104
P38
+ NDK173
c.ext193
medium227
gene expr 7 days
37.6
3378.5
1
0.84
7 days
cell extract
Fold change
C 3 gene expression in HFA by HIV
Sheet9
MVP[5pgP24/cellIL1-beta[10ng/ml]control extractmedium
37.63378.510.84
MVP[5pgP24/cell]IL1-beta[10ng/ml]control extractmedium
8.6812.310.6
Sheet8
1.0430.1891.0150.545
C3 gene exp 7days
4.84
37.6
1
376.7
3378.6
7 days
cell extract
fold change
C3 gene expression by HIV {MVP} in HFA on 7 days of infection
Sheet7
7days
MVP [J]1pg5pgc.extTNFIL1
4.8437.61376.73378.6
Sheet4
12negative16020U/ml13720ng/ml138
10720012610U/ml11910ng/ml119
601001250.2U/ml1382ng/ml137
5650
Sheet4
0
0
0
0
-/+HIV
Ratio pGLC3pro/RLTK
Induction of C3 promoter by HIV dose dependent
C3 gene expr march24
0
0
0
Jim's stock
Il 1-beta [U/ml]
Ratio pGLC3 pro/RLTK
Influence of IL1-beta on C3 induction promoter
Sheet13
0
0
0
New Il1-beta
IL1-beta [ng/ml]
Ratio pGLC3pro/RLTK
Influence of Il1-beta on C3 induction promoter
p50 NFkB BR37
1.68
2.82
2.44
3.67
1
1.04
24h
Cell extract
Fold change
C3 gene expresion in HFA by HIV
p50-p65p
Panomic dec12
0
00.2
00.3
00.35
00.53
00.027
00.024
00.545
0-0.01
cell extract
OD [p50-p65]
Detection of p50 subunits of heterodimer p50-p65 NFkB in HFA after HIV infection
Sheet12
0.2
0.3
0.35
0.53
0.027
0.024
0.545
-0.01
OD [NFkBp50]
Sheet6
0.20.0529150262
0.30.05
0.350.0264575131
0.530.0057735027
0.0270.001
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
**
***
****
****
****
cell extract
OD [NFkB subunit p50]
Sheet5
HIV [2pg]HIV [4pg]TNF[10U/ml]IL-1[5ng/ml]control extmediumpositive controlcompetition control
av0.20.30.350.530.0270.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333
SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503
Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007
NDK
Sheet3
NDK fNDK nNL4-3 fIL/TNFc.ext 10ulmedium
1.682.822.443.6711.04
3.63.491
1.60.7614
5081
67120
6040
116.665
67.7
0
0
0
0
0
0
0
Cape exp
inhibitor/+-IL1beta/+-HIV
Ratio pGLC3pro/RLTK
CAPE/+-HIV/+-IL1beta
0
0
0
0
0
0
GENISTEIN exp
inhibitor/+-HIV/+_ IL1-beta
RATIO pGLC3pro/RLTK
GENISTEIN/+-HIV/+_IL1-beta
0
0
Controls- HFA brain28/3 infection NL4-3/PBL
HIV/-HIV
RATIO pGLC3pro/RLTK
149
2430
148.5184.4
119.25210.25
81153
52.2573.33
120206.66
14.6616.33
2741
96152.33
59.33125.33
71.6670
00
2438
00
00
00
00
00
00
00
Ratio
Effect of HIV on the activity of different C3 promoter constructs in HFA
00
00
00
00
00
00
00
Ratio
Effect of HIV on the activity of different C3 promoter constructs in HFA [systemA/2]
MBD0003B060.doc
MBD000020EA.xls
C3protein wykres or
141.66512.667.66.4
108.66457.73.28.4
10.3387.331.043.2
19.16191.6626.3
20.762371.0712.5
0.311.533
0.2410.5
C
3d
7d
supernatants
C3 Protein [ng/ml]
inhibitors 2b graph
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graph cont inhib 2b
1577.9917269723
62.52
113.6055512755
150.2385979883
48h
cell extract
Relative Luciferase Activity [RLU]
kontrole do inhib 2b
HIVmedmock cIL-1TNF-mediummock c
average31111515762.51115
25.51316156.5551316
40.011118158.170.531118
26.95911156.461.97911
average30.82111515762.51115
SD7.991726972323.60555127550.95393920147.778553850223.6055512755
Ttest0.03534436910.00000031680.0006591335
IL-1 betaTNF-alfamediummock
15762.51115
156.5551316
158.170.531118
156.461.97911
av15762.51115
SD0.95393920147.778553850223.6055512755
0.0006591335
inhibitors2b
HIVIL-1 betaTNF- alfamediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium
3115762.5111533.61311.51111.43733382137.6302825
7.99172697230.95393920147.778553850223.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111
19-Jul
HIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumCalphostin/HIVCalphostin/medium
av31111533.61311.51111.437332825
SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031356.55743852432.6457513111
controle do graph inhib2b wl
controle do graph inhib2b wl
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
wl graph 1 Elisa p50NFkB
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graphknock 5000vpC3 prom
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
wl graph ad 5000 C3 prom bz Il1
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
daneknockout 5000vp promotor C3
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
1 EL p50NFkB
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
EL p50 controls
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
mC3RNA 24h, 3d signal transd
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
inhib2A wlasc grapg bez Il1
317.9917269723
112
153.6055512755
30.2385979883
3.60.3569425724
130.1616580754
11.50.5
110.8857388629
11.42.1166010489
370.7110365087
330.4150803135
286.5574385243
252.6457513111
48h
cell extract
Relative Luciferase Activity [RLU]
graph Inhib 2A bez Il1
1570.9539392014
62.57.7785538502
112
153.6055512755
48h
cell extract
Relative Luciferase Activity [RLU]
C3 protein wykres
C3 protein wykres
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
inhibitors 2A
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
C3 proteimna graph or
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
C3 proteina
C3 proteina
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
1NFkB ELISA
471.5275252317
242.5166114784
221.443375673
4.90.1527525232
6.20.2645751311
4.250.2306512519
38.31.5275252317
7.10.3511884584
6.90.2
48h
cell extract
Relative Luciferase Activity [RLU]
Exp 02/05Ad 5000vp/cellAd-IkB/mockAd-IkB/HIV
Br52/2Adenovirus-IkB
systemHIV+mockmediumAdenovirus-IkBAd-IkB/mockAd-IkB/HIVAdenovirusAd/mockAd/HIV+
average4724224.256.24.96.97.138.3
14822213.946.64.76.57.236
2491918.54.46.14.96.36.837
34624214.26.54.66.16.539
average47.522.2520.6254.186.44.73333333336.36.833333333337.3333333333
SD1.52752523172.51661147841.4433756730.23065125190.26457513110.15275252320.20.35118845841.5275252317
Br52/2
systemHIV+mockmediumAd-IkB/HIV+Ad-IkB/mockAd-IkB/mediumAd/HIV+Ad/mockAd-medium
average4724224.96.24.2538.37.16.9
14822214.76.63.94367.26.5
2491918.54.96.14.4376.86.3
34624214.66.54.2396.56.1
average47.522.2520.6254.73333333336.44.1837.33333333336.83333333336.3
SD1.52752523172.51661147841.4433756730.15275252320.26457513110.23065125191.52752523170.35118845840.2
0.280.0115470054
0.370.0251661148
0.40.0057735027
0.530.0057735027
0.0260.0005773503
0.0240.0005773503
0.5450.0025166115
-0.010.0005773503
30min
nuclear extract
OD value [450nm]
systemcontrol+Competition cIL-1 betaIL-6HIV+mockmedium
average0.2170.0330.2040.1240.1560.0620.047
10.2170.2170.1240.160.060.047
20.2140.0360.2080.1210.150.0610.044
30.220.0330.2040.1250.1620.0640.049
average0.2170.03450.20966666670.12333333330.15733333330.06166666670.0466666667
SD0.0030.00212132030.00665832810.0020816660.00642910050.0020816660.0025166115
HIV [2pg]HIV [4pg]TNF[10U/ml]IL-1[5ng/ml]control extmediumpositive controlcompetition control
av0.20.30.350.530.0270.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333
SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503
Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007
HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control
av0.280.370.40.530.0260.0240.545-0.01
0.30.40.40.530.0260.0230.54-0.01
0.280.350.390.520.0250.0240.542-0.011
av0.28666666670.37333333330.39666666670.52666666670.02566666670.02366666670.5423333333-0.0103333333
SD0.01154700540.02516611480.00577350270.00577350270.00057735030.00057735030.00251661150.0005773503
Ttest0.00000255570.00001811070.00000000.00000000.00000000.0000002
cell signalling involved in the activation of C3 gene expression
HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium
24h1410.672.90.580.4931.170.96.30.630.69
13.70.90.592.40.550.512.61.280.85.80.90.77
15.21.10.712.20.610.522.91.41.15.70.860.82
av14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76
SD0.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852
test
3d15.540.6560.783.50.40.93.12.11.86.70.780.5
16.40.70.793.40.520.913.22.21.66.40.980.6
16.30.630.822.950.490.893.52.31.56.20.890.67
av16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59
SD0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375
SD
HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium
24h14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76
3d16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59
SD240.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852
SD3d0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375
14.316.08350.79372539330.47031904071
10.6620.93333333330.10.0353836120.1527525232
0.65666666670.79666666670.96333333330.06110100930.020816660.118462371
2.53.28333333333.33333333330.36055512750.29297326390.4509249753
0.580.470.75666666670.030.062449980.0585946528
0.50666666670.90.80.01527525230.010.1
2.83333333333.26666666674.10.20816659990.20816659990.1732050808
1.28333333332.22.46666666670.11503622620.10.2516611478
0.93333333331.63333333332.09333333330.15275252320.15275252320.1101514109
5.93333333336.433333333311.40.32145502540.25166114781.1532562595
0.79666666670.88333333330.91333333330.145716620.1001665282.74
0.760.590.13233333330.06557438520.08544003750.0924463809
24h
3d
7d
cell extract
Relative quantification; Fold change
Cell signalling involved in the activation of C3 gene expression
0.79372539330.4703190407
0.10.035383612
0.06110100930.02081666
0.36055512750.2929732639
0.030.06244998
0.01527525230.01
0.20816659990.2081665999
0.11503622620.1
0.15275252320.1527525232
0.32145502540.2516611478
0.145716620.100166528
0.06557438520.0854400375
24h
3d
cell extract
Fold change [C3 mRNA]
135.33333333336.1101009266
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Re;ative Luciferase Activity [RLU]
135.33333333336.1101009266
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Re;ative Luciferase Activity [RLU]
141.66512.66
108.66457.7
10.3387.33
19.16191.66
20.76237
0.311.5
0.2410.5
3d
7d
supernatants
C3 Protein [ng/ml]
141.66512.667.66.4
108.66457.73.28.4
10.3387.331.043.2
19.16191.6626.3
20.762371.0712.5
0.311.533
0.2410.5
C
3d
7d
supernatants
C3 Protein [ng/ml]
HIVmockmediumCape/HIVCape/mockCape/mediumP38/HIVP38/mockP38/mediumSB580/HIVSB580/mockSB580/mediumSB190/HIVSB190/mockSB580/mediumAG17/HIVAG17/mockAG17/mediumJNK/HIVJNK/mockJNK/mediumGenistein/HIVGenistein/mockGenistein/medium
135.33333333336566.33333333335.56666666675.55.5333333333100.333333333311185.333333333333.333333333337.666666666733.33333333332121.666666666721.666666666753.666666666751.66666666674965.666666666747.3333333333493.66666666675.33333333333.6666666667Average
6.11010092664.35889894351.52752523170.51316014390.50.923760430717.6162803498.18535277198.96288643984.93288286238.504900548110.40832999734.35889894358.96288643988.08290376875.50757054736.65832811855.291502622111.01514109462.51661147842.64575131110.57735026920.57735026920.5773502692SD
0.00008431090.00001958870.00001947480.00002075310.02798383280.00100840240.024151360.00113383070.00774020590.00827556490.00024601170.00122031550.04907193280.01557401090.00690512192.51661147840.0055620.00001740970.00001942550.0000174097Ttest
135.33333333336.1101009266
37916.8226038413
654.3588989435
66.33333333331.5275252317
5.56666666670.5131601439
5.50.5
5.53333333330.9237604307
100.333333333317.616280349
1118.1853527719
85.33333333338.9628864398
33.33333333334.9328828623
37.66666666678.5049005481
33.333333333310.4083299973
214.3588989435
21.66666666678.9628864398
21.66666666678.0829037687
53.66666666675.5075705473
51.66666666676.6583281185
495.2915026221
65.666666666711.0151410946
47.33333333332.5166114784
492.6457513111
3.66666666670.5773502692
5.33333333330.5773502692
3.66666666670.5773502692
48h
cell extract
Relative Luciferase Activity [RLU]
141.66512.66
108.66457.7
10.3387.33
19.16191.66
20.76237
0.311.5
0.2410.5
3d
7d
supernatants
C3 Protein [ng/ml]
PROTEINAVERAGESD
IL-1141.66666677.637626158
TNF-alfa108.66666673.214550254
HIV [0.5]10.333333331.040833
HIV [1pg]19.166666672.020725942
HIV [2.5pg]20.766666671.078579312
mock0.30.2
medium0.2433333330.049328829
IL-1512.66666676.429100507
TNF-alfa457.76666678.494900431
HIV [0.5]87.333333333.214550254
HIV [1pg]191.66333336.345079458
HIV [2.5pg]236.666666712.58305739
mock3.510.5
medium30.1
IL-1 betaTNF-alfaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium
AV3d141.66108.6610.3319.1620.760.30.24
Av7d512.66457.787.33191.6623711.510.5
SD3d7.633.21.042.021.070.20.04
SD7d6.428.43.26.312.53.52
HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control
0.20.30.350.530.0270.0240.545-0.01
0.220.320.360.550.0230.0220.546-0.011
0.210.330.3670.60.0210.0230.546-0.012
av
SD
MBD0009EEC5.xls
22,3
53
24
50
5.65
2.5
1.4
0.5
25
22
22.5
1-control(+)HIV, 2-Control(-)HIV, 3-Calphostin-C, 4-SQ22536, 5-CAPE 1:10000, 6-CAPE 1:15000, 7-Genistein 1:1000, 8-Genistein 1:2000, 9-PMA, 10-Pertusis toxin, 11-8Br-cAMP
inhibitors
RATIO pGLC3pro/pRLTK dual luciferase assay
Br22/3 C3 PROMOTER INDUCTION by HIV with INHIBITORS
22,3+-
5312
2421
5033
5.654.5
2.54.6
1.40.98
0.50.4
2522
2215
22.515
contr(+)/(-)
Calph
SQ22536
CAPE:10000
CAPE;15000
Genist:1000
Genist:2000
PMA
Pertusis toxin
8Br-cAMP
+HIV
-HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK Dual Luciferase Assay
BRAIN 22/3
Sheet1
5312
2421
5033
5.654.5
2.54.6
1.40.98
0.50.4
2522
2215
22.515
Sheet1
0
0
0
0
0
0
0
0
0
0
0
inhibitors(-)HIV
inhibitors
Ratio pGlC3/pRLTH
C3 induction promoter - inhibitors without HIV
Br22,4
Br22,4
49
25
16.5
44
18
7.6
4.7
1.2
20
22
31.4
35
Br22/4 inhibitors (+) HIV
inhibitors
Ratio pGL3C/pRLTK
-
20
22.5
30
33.5
11
9.7
1.3
3.4
20
20
32
25
inhibitors(+) HIV
inhibitors
Ratio pGLC3/pRLTK Dual-Luciferase Assay
BR 22/4 C3 induction promoter
49
25
16.5
44
18
7.6
4.7
1.2
20
22
31.4
35
inhibitors (-) HIV
18,4
50
46
26
42
18.4
126
84
72.5
75.6
inhibitors (+) HIV
inhibitors
Ratio pGLC3/pRLTK
Br 18/5 C3 induction promoter
18,4 bez HIV
23
42
35
56
16
72
45
80
38
inhibitors (-) HIV
br4p4
4920
2522.5
16.530
4433.5
1811
7.69.7
4.71.3
1.23.4
2020
2220
31.432
3525
control(+)/(-)
Calph:1000
Calph10-5
SQ22536
CAPE:20000
CAPE:15000
Genist:2000
Genist:1000
8-Br-cAMP(1pgHIV)
PMA(1pgHIV)
Pertusis t1x
Pertusis t5x
(+)HIV
(-)HIV
inhibitors with/w-o HIV
RATIO pGLC3pro/pRLTK
Brain 22/4
C 3 promoter induction March06
7.5
4.5
2.5
3.8
1
cell extract
Fold change [Relative Luciferase Unit
C3 promoter induction by HIV in HFA
MVP and C3promoter
7.5
4.5
2.5
3.8
1
cell extract
Fold change [Luciferase Activity Unit]
C3 promoter induction by HIV[MVP] in HFA
Sheet14
IL1-beta