www.sciencemag.org/cgi/content/full/330/6002/390/DC1

Supporting Online Material for

Salmonella Pathogenesis and Processing of Secreted Effectors by Caspase-3

C. V. Srikanth, Daniel M. Wall, Ana Maldonado-Contreras, Haining Shi, Daoguo Zhou, Zachary Demma, Karen L. Mumy, Beth A. McCormick*

*To whom correspondence should be addressed. E-mail:

Beth.McCormick@umassmed.edu

Published 15 October 2010, Science 330, 390 (2005) DOI: 10.1126/science.1194598

This PDF file includes:

Materials and Methods Figs. S1 to S6 Tables S1 and S2 References

1

Supporting online material for

Salmonella pathogenesis and processing of secreted effectors by caspase-3

C.V. Srikanth1,2*, Daniel M. Wall1,3,*, Ana Maldonado-Contreras2, Haining Shi1, Daoguo Zhou 4,Zachary Demma 2, Karen L. Mumy1,2, and Beth A. McCormick1,2

1 Department of Pediatric Gastroenterology and Nutrition, Harvard Medical School and

Massachusetts General Hospital, Boston, MA 02129, USA.

2 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical

School, 55 Lake Avenue North, Worcester, MA 01655, USA.

3 Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life

Sciences, University of Glasgow, G12 8QQ, UK.

4 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

* Contributed equally to this work

To whom corresponding should be addressed:

E-mail: Beth.McCormick@umassmed.edu

2

Materials and Methods

Tissue culture:

T84 intestinal epithelial cells (American Type Culture Collection, Rockville, Maryland)

(Passages 50-70) were grown in a mixture of Dulbecco’s Modified Eagles Medium and Ham’s

F12 Nutrient mixture supplemented with 14 mM NaHCO3, 15 mM Hepes buffer (pH 7.5), 40

mg/liter penicillin, 8 mg/liter ampicillin, 90 mg/liter streptomycin, and 5% fetal bovine serum.

T84 cell monolayers were grown and maintained on inverted 0.33-cm2 ring-supported, collagen-

coated polycarbonate filters (cell culture inserts) (Costar Corp., Cambridge, MA), as previously

described (1) with modifications (2). Steady state transepithelial resistance (TER) was measured

using Millipore Millicell- TERS with “chopstick” electrodes (World Precision Instruments,

Sarasota, FL). A confluent monolayer corresponded to 400-1500 cells/cm2, consistent with

measurements by Dharmsathaphorn and Madara (1).

Bacterial strains, plasmids and growth conditions:

Strains and plasmids used in this study have been previously described (3). All strains were

grown in Luria broth (LB) as previously described (4). Strains carrying pBH (Roche,

Indianapolis, IN) or its derivatives were cultured in media containing 50 µg/ml ampicillin.

Construction of the caspase site mutant proteins:

The aspartic acid to alanine mutation (DEVD to DEVA in SipA) was performed using the

QuickChange site directed Mutagenesis kit (Stratagene, La Jolla, CA). The pBH-SipA was used

as a template together with the primer pair (Cas3F:

3

5’CTTTTGATGAAGTCGCTGGCGTAACCAGC and Cas3R: 5’

GCTGGTTACGCCAGCGACTTCATCAAAAG) in the mutagenic PCR, according to the

manufacturer’s instructions. The resulting mutants were sequenced to confirm the presence of

the desired mutation. SopA expressing plasmids (pSopA: pZP188) were used as a template to

generate pcsm-SopA (plasmids encoding caspase-3 sites mutant SopA ) using oligos

SopA_mutF: GTGTTGCTGGATTCGTTTGCCAGGAACAGCGCCGCCATG,

SopA_mutR: CATGGCGGCGCTGTTCCTGGCAAACGAATCCAGCAACAC in the 1st

mutagenic PCR. The resulting mutant plasmid was used as a template for a second round of

mutagenesis with the oligo pair SopA_mut_b_F (1448 A-C):

GATTTTACACTGTATGACAGGCGTGGCCTGCACTGATGGCACCCGCCAGAAAGC

SopA_mut_b_R (1448 A-C):

GCTTTCTGGCGGGTGCCATCAGTGCAGGCCACGCCTGTCATACAGTGTAAAA in the

2nd mutagenic PCR. The resultant mutant plasmids were confirmed for the desired mutations by

sequencing.

SDS-PAGE and Western blotting:

Samples were separated through an 8-16 % gradient Tris-HCl Ready Gel (Biorad, Hercules, CA)

by polyacrylamide electrophoresis (SDS-PAGE) and transferred to nitrocellulose. Immunoblots

were performed using a 1:5,000 dilution of murine anti-HA monoclonal antibodies (Covance,

Richmond, CA). Immunoblots for caspase detection were carried out using 1:5,000 dilutions of

antibodies specific for caspase-1, caspase-3, caspase-8 and activated caspase-3 (Cell Signaling

Technology, Danvers, MA). Goat anti-rabbit and goat anti-mouse IgG labeled with horseradish

peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) and diluted 1:2,000 were used to detect

4

the bands, which were visualized by enhanced chemiluminescence using the Super Signal ECL

kit (Pierce, Rockford, IL). Samples were also electrophoresed on separate gels and Coomassie

stained to confirm equal protein loading between samples. For native SipA detection, SipA

monoclonal antibodies were used (gift from Dr. Eduourd Galyov, University of Leicester, UK)

and for the detection of HA-tagged SipA, anti-HA antibodies were purchased from Cell

Signaling (Danvers, MA). For SopA western blots affinity purified anti-SopA antibodies

generated in Rabbit were used at 1:400 dilution.

PMN transepithelial migration assay:

The physiologically directed (basolateral to apical) PMN transepithelial migration in response to

bacterial stimulus has been previously described in detail (5). Briefly, human peripheral PMNs

were isolated from anti-coagulated human whole blood by a gelatin sedimentation technique as

previously described (6). T84 cells were stimulated at the apical surface with S. enterica serovar

Typhimurium, S. Dublin or E. coli F-18 at a multiplicity of infection (MOI) of 375 for 1 hour.

Formyl-Methionyl-Leucyl-Phenylalanine (fMLP), a neutrophil chemoattractant, was used as a

positive control in all assays. Following extensive washing, neutrophils (PMN; 1 x 106) were

added to the basolateral surface and allowed to transmigrate through the monolayer for 2 hour.

PMN transmigration was quantified by assaying for the PMN specific azurophilic granule

marker myeloperoxidase (MPO), as previously described (6). PMN cell equivalents were

estimated from daily PMN standards as the number of PMN that had completely traversed the

monolayer into the apical chamber.

5

Mouse infections

Six-eight week old female C57BL/6 mice, or C57BL/6 caspase-3 knock out mice were infected

using a modified version of the streptomycin pre-treatment model (7). Water and food were

withdrawn 4 hour prior to treatment with 3.75 mg of streptomycin intragastrically. The following

day, water and food were again withdrawn for 4 hour before the mice were infected with 5 x 107

CFU of S. Dublin (500 µl of a suspension in PBS intragastrically by gavage). S. Dublin was not

induced with IPTG as this strain produces the proteins constitutively. Mice were left for 48 hour

before sacrificing and removal of the colon and cecum for examination. Care of animals is in

accordance with institutional guidelines under protocol number: 1993-08.

Histological procedures:

Proximal colon samples were placed in Tissue Tek optimal cutting temperature compound

(OCT) (Miles, Inc., Elkhart, IN) and snap frozen in liquid nitrogen. Cryosections (5 µm) were

mounted on glass slides before either hematoxylin and eosin (H&E) or fluorescent staining. H&E

staining was carried as previously described (8). Fluorescent staining was carried as follows:

Slides were fixed in acetone on ice for 10 minutes before submerging in a 1% bovine serum

albumin (BSA) solution. Slides were blocked by adding an avidin solution in 1% BSA for 15

minutes, washed, and then biotin in 1 % BSA was added for a further 15 minutes (Vector

Laboratories, Burlingame, CA). Sections were stained using a fluorescein-iso-thiocyanate (FITC)

labeled anti mouse Ly-6G and Ly-6C (Gr-1) antibody (BD Biosciences Pharmingen, Franklin

Lakes, NJ) for neutrophils (PMN). After washing, DNA was stained and mounted using the 4’

6’-diamidino-2-phenylindol (DAPI) containing solution Vectashield (Vector Laboratories,

Burlingame, CA).

6

Histopathological examinations:

At necropsy, colonic tissues were collected and frozen in OCT compound prior to storing at

_80°C. Five-_m sections were cut on a 2800 Frigocut cryostat (Reichert-Jung, Germany) and

were stained with hematoxylin and eosin (H&E). Three tissue samples from the proximal,

middle, and distal parts of the colon were prepared. The sections were analyzed without prior

knowledge of the type of treatment. Colonic pathology was scored by using a modified histology

scoring system based on previously published methods (9, 10). The scoring system consists of

two parts. Part 1 is the determination of the infiltration of inflammatory cells in the colon, with

scores ranging from 0 to 4 (0, normal cell pattern; 1, scattered inflammatory cells in the lamina

propria; 2, increased numbers of inflammatory cells in the lamina propria; 3, confluence of

inflammatory cells extending into the submucosa; and 4, transmural extension of the infiltrative

inflammatory cells). Part 2 is the evaluation of colon tissue damage, with scores that also range

from 0 to 4 (0, normal tissue pattern; 1, minimal inflammation and colonic crypt hyperplasia; 2,

mild colonic crypt hyperplasia with or without focal invasion of epithelium; 3, obvious colonic

crypt hyperplasia, invasion of epithelium, and goblet cell depletion; and 4, extensive mucosal

damage and extension through deeper structures of the bowel wall). The total colon pathology

score equals the inflammatory cell score plus the tissue damage score.

Myeloperoxidase assay:

Myeloperoxidase (MPO) activity was measured using the Mouse MPO ELISA test kit from Cell

Sciences (Canton, MA). Briefly, sections were taken from the proximal colon and snap frozen in

liquid nitrogen until testing. Upon thawing, the sections were weighed and then homogenized in

7

lysis buffer as specified by the manufacturer. An ELISA assay was carried out as per

manufacturer’s instructions and the amount of MPO per section was expressed as ng of MPO per

gram of tissue.

RNA analysis:

Total RNA was isolated from control or infected cells at various time points post infection using

TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA

was DNase treated prior to reverse transcription with random hexamers using a GeneAmp Kit

(Roche, Indianapolis, IN). Quantitative real time PCR analysis of caspase-3 and GAPDH mRNA

levels was performed using a SYBR Green master mix (Applied Biosystems, Foster City, CA)

with the following primers; Cas-3For 5’-CAAACTTTTTCAGAGGGGATCG-3’, Cas-3Rev 5’-

GCATACTGTTTCAGCATGGCAC-3’, GAPDH For 5’-AGAACATCATCCCTGCCTC-3’ and

GAPDH Rev 5’-GCCAAATTCGTTGTCATACC-3’. Amplification was carried out on an

iCycler (Bio Rad, Hercules, CA) and the data analyzed using Opticon Monitor 2 software

(Promega, Madison, WI). The amount of caspase-3 transcript was normalized to the amount of

GAPDH transcript and the normalized values of the Salmonella-infected cells versus the control

cells were used to calculate the relative expression using the delta delta Ct method.

Apoptosis Assay (Annexin V): T84 cells grown on 6 well transwell plates (Costar) were either

treated with the apoptosis inducer staurosporine (1mg/ml) or infected with wild-type S.

Typhimurium (SL1344) for 3 hour at 37 ºC. The plates were subsequently treated with 5 mM

EDTA for 10 minutes at 37 °C. Cells were then removed from the plates with cold PBS and

approximately 2 x 106 cells from each sample were prepared for the FACS analysis using the

8

Annexin V-FITC apoptosis detection kit (Sigma Chemical Co., St. Louis, MO) following the

manufacturers protocol.

Generation of small interfering RNAs (siRNA) for suppression of caspase-3 expression:

The plasmid used to generate siRNAs was constructed using the pSUPER vector (Oligoengine,

Seattle, WA), in the method described by Brummelkamp et al (11). Oligonucleotides were

designed incorporating a 19-nt sequence (in italics) from the human gene encoding caspase-3

(Genbank Accession number NM_032991), its reverse complement (in italics) separated by a

short spacer region, and BglII or HindIII restriction sites. For caspase-3: 5’-

GATCCCCTCAATGGACTCTGGAATATTTCAAGAGAATATTCCAGAGTCCATTGATTTTTGGAAA-3’ and 5’-

AGCTTTTCCAAAAATCAATGGACTCTGGAATATTCTCTTGAAATATTCCAGAGTCCATTGAGGG-3’; and for a

random control sequence, 5’-

GATCCCCCCGACAAGCTTGAATTTATTTCAAGAGAATAAATTCAAGCTTGTCGGTTTTTGGAAA-3’ and 5’-

AGCTTTTCCAAAAACCGACAAGCTTGAATTTATTCTCTTGAAATAAATTCAAGCTTGTCGGGGG-3’.

Oligonucleotides were annealed, yielding double-stranded DNAs with overhanging restriction

sites, and ligated into digested pSUPER. Constructs were transformed into competent E. coli

DH5α by standard methods, and plated on LB with ampicillin (50 µg/ml). Plasmids were

extracted (QIAprep Spin Mini-prep Kit, Qiagen, Valencia, CA) and sequenced for confirmation.

Once confirmed, bulk plasmid was prepared for transfection using Qiagen Plasmid Midi Kit

(Qiagen, Valencia, CA).

9

Transfection of HCT8 intestinal epithelial cells:

HCT8 cells (kind gift of Cheleste Thorpe, Tufts University School of Medicine) are a polarizing

intestinal human transformed cell line and were used for this protocol because of their high

transfection efficiency. HCT8 cells were transfected with the modified pSUPER using

Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per manufacturer’s instructions. Briefly, 4 µg of

plasmid was diluted into RPMI 1640 without serum (Invitrogen, Carlsbad, CA). Separately,

Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was diluted into RPMI 1640 with Fetal Bovine

Serum (FBS) and incubated at room temperature for 5 minutes. Following incubation, the two

mixtures were combined and incubated at room temperature for 20 minutes. This cocktail was

added to HCT8 cells with RPMI 1640, and the cells were incubated in RPMI with 8% v/v FBS

without selection. Cells were passaged into fresh media with selection added the next day

(neomycin-G418, 1 mg/mL; Sigma-Aldrich, St. Louis, MO). Cells underwent two additional

cycles of growth/passage in G418 prior to use.

Functional assay for the caspase-1 and caspase-3:

Mucus samples from infected and uninfected mice were extracted by very gentle scraping using

a rubber policeman, as previously described (12). To determine caspase-1 and caspase-3 activity,

the mucus extract was directly mixed with the assay buffer according to the manufacturer’s

instructions (BioVision, Mountain View, CA).

Determination of SipA and SopA processing/cleavage:

In vitro cleavage of SipA and SopA was performed using a recombinant caspase-3 enzyme (20

µg/ml; Axxora, San Diego, CA). This enzyme was directly added to secretory protein extracts

10

(obtained by precipitation of bacterial culture supernatant proteins using 15% TCA precipitation)

of bacteria expressing these proteins and their caspase-3 site mutant forms for an hour at 37oC.

The samples were then immunoblotted with antibodies against SipA or SopA to verify cleavage.

Structural prediction of SopA caspase-3 sites

Software PYMOL (Protein molecule view and edit tool; DeLano Scientific, LLC) was used to

predict and view the three-dimensional structural positioning of the caspase-3 sites of SopA

using the deposited protein structure (ID 2QYU).

Statistics:

Neutrophil isolation was limited to repetitive donations by five different donors over the course

of these experiments. Due to variations in both neutrophils and transepithelial resistance between

monolayers (baseline resistance between 400 and 1,500 Ωcm2), data were analyzed within an

individual experiment and not between experiments. However, the overall trends associated with

these data are reproducible between experiments. All results are expressed as the mean ±

standard deviation of an individual experiment done in triplicate. P values were calculated

according to Student’s t test, and values <0.05 were considered statistically significant.

Invasion assay in primary macrophages

Bone marrow from freshly dissected wild-type and caspase-3 KO mice were collected in

conditioned differential media for bone marrow culture (RPMI with conditioned serum). The

cells were allowed to grow for 5-7 days in Petri plates. Using a 0.02% EDTA solution the cells

were scraped and after normalizing the cell number about 200,000 cells per well of a 24 well

plate. After overnight incubation these cells were washed and infected at a multiplicity of

11

infection of 10 with wild-type Salmonella. The cells were washed and fresh buffer containing

100 microgram /microliter of gentamycin was added. Bacterial cfu were determined 4 hours post

infection.

S1

0 1 2 3 4

SL1344

A-Casp3

Casp3

Hours following infection

A

0 1 2 3 4

vv341

Hours following infection

A-Casp3

Casp3

B

A1/ SipA A1/ csm

A

B

C

S2

* *

ΔSipA/pCSM-SipAΔSipA/pSipA

ΔSipA/pCSM-SipAΔSipA/pSipAΔSipA

S3

SL1344 fMLP HBSS+05

1015202530354045

PMN

Mig

ratio

n (C

E x

10,0

00)

UntreatedCasp3

inhibitor (20 µM)

*

Caspase-1 inhibitor

0

5

10

15

20

25

30

SL13442 20 0

HBSS2 20 0

fMLP2 200

PMN

Mig

ratio

n (C

E x

10,0

00)

A B

(µM)

S4

0

0.2

0.4

0.6

0.8

1

1.2

15 min 30 min 60 min 120 minTime Post Infection

Rel

ativ

e Ex

pres

sion

S5

SL1344 fMLP HBSS+05

1015202530354045

PMN

Mig

ratio

n (C

E x

10,0

00)

*

Untreated

Caspase 3(20 µM)

Caspase 1(20 µM)

0.2

0.4

0.6

0.8

1

1.2

1.4

0.5 1 1.5 2 3 4

Time post infection (h)

LDH

Act

ivity

(uni

ts/m

l)

SL1344SipA-HilA-HBSS+Triton

S6

60- NVWARDYLDLAQNK -73381- KGAGLDIGDNTTVD -394666- TKVHIDMYDGKNIT -679663- FGFRQDIKDGVSVS -676

1032- NKRMGDLRDTQGDA -10451075- RKHELDGVDLFTGA -1088

EspF * None detectedEspG * 157- ALSKMDLTDHKSMH -170EspH * 27- HIPNEDIVDEIQLK -37EspZ None detectedMap * None detected

EspA 2 sites non-conserved among strains

156- SEGIADAADDAAG -169 a

181- ASRTSDVADDIADSAQRA -198 b

EspD None detected

281- SIIDVDANDLQQLG -294410- RLLLDIQDGNQSTN -418

EscD 381- VIDAIDPKDGINIS -394EscF None detectedEscJ None detectedEscN None detectedEscR None detectedEscT 241- ANIHSDIIDRSLPS -254EscU None detectedEscV 191- ILLHHDAGDIIYSF -204SepL None detectedSepQ 257- NAKKDDIIDSTGYH -270SepZ None detected

5- FSSLQDIYDFYQDG -1826- NLTQQDLNDLHSYA -39

EspB*

CesD

Esc/Sep T3SS

Chaperone

EscC

Enteropathogenic Escherichia coli (EPEC)

EspC

Protein translocation apparatus

LEE-effectors protein

Table S1. Caspase-3 recognition and cleavage sites in Enteropathogenic Escherichia coli secreted effectors.

a Human Enteropathogenic strainE2348/69; Serotype O127 : H6(Accession number: CAA79733)b Rabbit diarrheagenic Escherichiacoli RDEC-1; serotype 015:H(Accession number: AAK26729)

* Proteins reported having differentfunctions related to the N and C-terminus or specific protein region

Analysis of the primary acid amino sequence.The caspase-3 motifs are highlighted in red.

203- SLKLDDLVDAQAKLA -217258- NDILEDIKDSHPIQ -270

275- PTPPEDMPDGGPTP -288386- FNHTPDNSDGIGSD -399553- TETNADTIDKNHAI -566590- ELLTDDISDLKNNN -603

IpaB None detectedIpaC* 270- DISLQDKIDTQRRT -283IpaD None detectedIpaJ None detected

OspB 49- EKGFCDSNDVLSIM -6212- TQCNIDSMDNRLHT -25138- HQSNSDLSDKILNI -151221- YMTLTDHFDNAIPP -234

OspC2 12- VQNNIDLVDGKINP -25OspF None detected

49- AEIFEDVNDSSALY -62103- PLSDIDTADIPDNI -116

MxiA None detected20- AMTNSDDGDETADA -3341- NSKYIDSSDEMASA -54

101- LPLDQDFIDRLKRY -114112- KRYFKDPSDQVLAL -125

MxiD * None detectedMxiE 54- FYHENDLRDSCNTE -67

MxiG * None detectedMxiH None detectedMxiI None detected

MxiJ * None detectedMxiK None detectedMxiL None detected

MxiM * None detectedMxiN None detectedSpa9 None detected

Spa13 None detectedSpa15 None detectedSpa24 None detectedSpa29 None detectedSpa32 21- KLSSIDNIDSLVLK -34Spa33 None detectedSpa40 None detectedSpa47 305- LLEDDDFADPLAEE -318IpgD None detected

VirB None detected

IpgC 29- NAIPDDMMDDIYSY -421- MEDLADVICRA -11

13- GIPSIDTDDQAIML -26

Mxi/Spa secreted proteins

OspC1

OspG

IpaA*

Shigella flexneri

Transcriptional activator

Chaperone

IpgE

Mxi/Spa T3SS

MxiC *

Table S2. Caspase-3 recognition andcleavage sites in Enteropathogenic Shigellaflexneri secreted effectors.

* Proteins reported having differentfunctions related to the N and C-terminus or specific protein region

Analysis of the primary acid amino sequence. Thecaspase-3 motifs are highlighted in red.

Figure Legends to Supplemental Data

Fig. S1. Time course of caspase-3 activation. T84 cells were infected with either wild-

type S. Typhimurium (SL1344) or an isogenic invasion deficient strain (vv341) over a

four-hour time course. Immunoblots were performed with anti-caspase-3 antibody (Cell

Signaling Technology). A-Casp3 represents the activated form of the enzyme.

Fig. S2. Analysis of PMN infiltration. A) PMN infiltration into the proximal colon was

quantified by tissue myeloperoxidase activity (MPO) in mice following a 48 hour

infection with the ∆SipA, ∆SipA/pSipA, and ∆SipA/pCSM-SipA strains. MPO activity

was measured using the Mouse MPO ELISA test kit from Cell Sciences. Data are

expressed as the mean ± SD and represent groups of five mice. (*), P < 0.01 when the

data set for a given infection was compared to the wild-type (SL1344) S. Typhimurium.

B) Fluorescently stained proximal colon sections at 20x magnification. Sections were

stained with DAPI (blue) and PMNs were stained using a FITC-labeled antibody (green)

specific for the PMN surface markers Ly-6G and Ly-6C. Shown is a comparison between

the SipA complemented strain (∆SipA/pSipA) and the caspase-3 site mutant

complemented strain (∆SipA/pCSM-SipA). (C) Bone-marrow-derived macrophages from

wild-type and caspase-3-/- (caspase-3 KO) mice were infected (at a multiplicity of

infection of 10) and the bacterial colony forming units (cfu) were determined 4 hour post

infection.

Fig. S3. Pharmacologic inhibition of caspase-3. A) The effect of the specific caspase-3

inhibitor on S . Typhimurium-induced PMN transepithelial migration across T84

monolayers. Prior to infection, T84 monolayers were exposed to buffer only (black bars),

or the caspase-3 inhibitor for 2 hour. (B) The effect of the specific caspase-1 inhibitor on

S. Typhimurium-induced PMN transepithelial migration across T84 monolayers. PMN

migration across uninfected T84 monolayers in the presence of 1 µM PMN

chemoattractant (fMLP), or buffer served as the positive and negative controls,

respectively. The data are expressed as the mean ± SD of triplicate samples and represent

one of at least three independent experiments performed with similar results. (CE), cell

equivalents, (*), P < 0.01.

Fig. S4. Quantitative RT-PCR analysis of caspase-3 expression. Caspase-3 mRNA

production in response to S. Typhimurium infection was monitored over the first 2 hour

of infection. Caspase-3 mRNA levels were normalized to GAPDH transcript levels and

the normalized values of the Salmonella-infected versus the control uninfected cells were

used to calculate and plot caspase-3 mRNA relative expression. The data are expressed as

the mean ± SD of triplicate samples and represent one of at least three independent

experiments performed with similar results.

Fig. S5. The effect of the exogenous addition of caspase-3 on S. Typhimurium-induced

PMN transepithelial migration across T84 monolayers. During S. Typhimurium wild-type

infection, T84 monolayers were exposed to buffer (black bars), caspase-3 (20 µM; white

bars), or caspase-1 (20 µM; gray bars) for 1 hour. PMN migration across uninfected T84

monolayers in the presence of 1 µM PMN chemoattractant (fMLP), or buffer only served

as the positive and negative controls, respectively. The data are expressed as the mean ±

SD of triplicate samples and represent one of at least three independent experiments

performed with similar results. (CE), cell equivalents, (*), P < 0.05.

Fig. S6: Cell lysis in response to infection with S. Typhimurium. Lactate dehydrogenase

(LDH) activity in the supernatant of T84 infected cells was measured over 4 hour as an

indicator of cell lysis in response to infection. Wild-type S. Typhimurium (SL1344) and

the SipA negative mutant strain SipA- and HilA-, an invasion deficient strain, were used

to infect T84 cells for 1 hour and LDH release was quantified using an In Vitro Toxicity

Assay kit (Sigma-Aldrich). One percent Triton and HBSS+ buffer were used as positive

and negative controls, respectively. The data are expressed as the mean ± SD of triplicate

samples and represent one of at least three independent experiments performed with

similar results.

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