www.pnas.org/cgi/doi/10.1073/pnas.1813458115

2

Lee et al. Supplementation information Supplemental Materials and Methods

Materials. DMEM, RPMI 1640, penicillin/streptomycin, and trypsin-EDTA were purchased

from Invitrogen. Sheep EA’s targets and GVB++ buffer were purchased from Berlex.

Versene was obtained from Lonza. LPS was purchased from Sigma (Escherichia coli

0127:B8). Rapamycin was purchased from Sigma. The Adenovirus expression vector for

mature SREBP-1a was described previously (1 ) .

Animals. The original SREBP-1a-deficinet mouse line (SREBP-1aDF) was described

previously ( 2 ) but as reported by Gerlic et al. (3) and Im et al. (4), the SREBP-1a gene

trap was inserted into the Srebf1 locus on chromosome 11 within the genome of B129 strain.

The Nlrp1 locus is tightly linked to the Srebf1 locus and after 10 generations of backcrossing

to C57Bl6J, the mice still had the Nlrp1 locus from B129. Because the Nlrp1a gene from

the B129 locus is not expressed for an unknown molecular reason these mice did not

express Nlrp1a. Knowing this limitation, we subjected the mice to several more rounds of

backcrossing to facilitate recombination to generate the SREBP-1aDF/B6 mice used in the

current study, which contains the Nlrp1 locus from C57Bl6J mice. Mice with floxed alleles

for raptor on C57BL6J background (5) and SCAP on a mixed B6/129 (6) were obtained from

JAX. The SCAP floxed mice were backcrossed with C57BL6/J mice for 12 generations. The

myeloid specific knockouts for both raptor and SCAP were generated by mating into the

C57BL6J strain of LysM6 CRE mice. Knockout and control littermate floxed mice were used

in all experiments. Mice were maintained in 12 h light/12 h dark cycles with free access to food

and water in pathogen free mouse facility. Mice were harvested for bone marrow isolation

at the end of the dark cycle. All procedures were performed in accordance with the

Institutional of Animal care and Use Committees at the Sanford Burnham Prebys Medical

Discovery Institute and the Keimyung University School of Medicine, Daegu, South Korea

(KM-2015-32R3).

Phagocytosis assay. Phagocytosis assays were performed using slight modifications to a

previously published procedure (8). Opsonized target particles for the phagocytosis assay

were sheep erythrocytes (E) with either IgG anti-sheep red blood cells (EAIgG) prepared as

2

previously described (7). Briefly, eight-well Lab Tek chambers (Nalge Nunc Intl., Naperville, IL)

were coated with human serum albumin (HSA) (MDI/American Red Cross, Louisville, KY) as

a control protein. Phagocytic cells resuspended in phagocytosis buffer (RPMI, supplemented

with 2 mM L-glutamine, 10 µg/ml pen/strep, 5 mM MgCl2) are added to each chamber, the

cells centrifuged at 700 rpm (RT6000, Dupont Sorvall) for 3 min and subsequently placed at

37 °C in 5% CO2 for 30 min. Targets were then added (106/100 µl), the slides again

subjected to centrifugation (700 rpm, 3 min), and incubated for 30 min at 37 °C. After

removing unbound targets by washing, bound, uningested targets were removed by

hypotonic lysis ( 8 ) (). Cells were then fixed in freshly diluted 1% glutaraldehyde and stained

with GIMSA (Sigma). Phagocytosis was quantitated using light microscopy. The number of

E-targets ingested per 200 effector cells was defined as the phagocytic index (PI), whereas

the percentage of effector cells ingesting at least one E-target was defined as the percent

phagocytosis. Over 200 effector cells were scored per well and duplicate sample wells per

condition were used for each experiment.

Isolation of BMDMs. Bone marrow-derived macrophages were isolated from C57B/6J,

SREBP-1aDF/B6, SCAPfl/fl, SCAP LysM6-SCAP KO (SCAP mKO), Raptorfl/fl, or LysM6-

Raptor KO (raptor mKO) mice. Femurs and tibias were isolated and flushed with DMEM

supplemented with 2% FBS. After RBC were lysed using ACK lysis buffer (0.15 M NH4Cl, 10

mM KHCO3, and 0.1 mM Na2-EDTA, pH 7.2), macrophages were depleted by a pre-

adhesion step for 2 h at 37°C in 5% CO2. Following the differentiation for 7 days in culture,

BMDMs were subcultured to use in experiments.

Adenovirus infection in BMDMs. Macrophages were plated in 100 mm dishes (1 x

106 cells/dish). Ad-SREBP-1a (10 multiplicities of infection [moi]) was added to cells in DMEM medium containing 20% L929 conditioned medium without FBS at 37°C with 5% CO2 for 6

h., and medium with FBS was added. After incubating for additional 24 h, cells were collected

for analysis.

Isolation of RNA and qPCR. Total RNA was prepared from macrophages of WT and

SREBP-1aDF/B6 mice by the Trizol procedure (Invitrogen) and cDNA was synthesized

using cDNA superscript kit (Bio-Rad) to use for qPCR with CFX96 Bio-Rad qPCR machine

3

(Bio-Rad). mRNA levels were normalized for expression of ribosomal protein L32 mRNA as

control and calculated by the comparative threshold cycle method.

Immunoblotting. Proteins were isolated from BMDM and immunoblotting was performed

following a modified protocol of previous method (9). Proteins were resolved by 5-10% Tris-

HCl SDS/PAGE gel electrophoresis and transferred onto nitrocellulose (Bio-Rad). Antibodies

to the following proteins were purchased from Cell Signaling Technology (Danvers, MA,

USA): Raptor (catalogue #2280), S6 (catalogue #2217), phosphor-S6 (catalogue # 2211),

p70S6K (catalogue #9202), phosphor-p70S6K (catalogue #9234), Moesin (catalogue #3142),

cell division control protein (CDC) 42 (catalogue #4651), Cofilin (catalogue #5175), Profilin-

1 (catalogue #3246), WASP-family verprolin-homologous protein (WAVE)-2 (catalogue

#3659), the ras-related C3 botulinum toxin substrate (RAC)1/2/3 (catalogue #2465), and actin-

related protein (ARP) 2 (catalogue #3128). SREBP-1 antibody (IGG2A4) was purified from

hybridoma CRL2121 that was obtained from ATCC. Anti-a-actin (catalogue #A5441) was

purchased from Sigma (Sigma-Aldrich, St. Louis, MO. USA). Proteins were resolved by SDS-

polyacrylamide gel electrophoresis, transferred to 0.2 mm nitrocellulose membranes (GE

Healthcare Amersham) by wet transfer at 4 °C, washed in Tris-buffered Saline containing

Tween 20 (TBST) for 30 min, blocked in 5% skim milk for 1 h at room temperature, and

washed for a further 30 min in TBST prior to incubation with primary antibodies. All antibodies

were used at a concentration of 1:1000, with the exception of a-actin which was used at

1:4000, and were made up in TBST containing 2.5% (wt/vol) BSA and 0.01% (wt/vol) NaN3.

After overnight incubation at 4 °C, membranes were washed for 50 min at room temperature

in TBST, and incubated with the appropriate secondary antibody for 1 h at room temperature.

Then membranes were washed for an additional 40 min in TBST at room temperature before

developing. All immunoblots were developed by incubation with horseradish peroxidase-linked

secondary antibodies at room temperature for 1 h. Protein bands were visualized using ECL

reagents (Bio-Rad Laboratories, Hercules, CA) and detected by chemiluminescence imaging

system (Fusion Fx, Vilber).

Bio-distribution and clearance of IC-IP6 from the blood. Preparation of IC-IP6, which

is phytic acid chelated with Fe3+ was prepared as described ( 1 0 ) . The fate of injected

IC-IP6 in vivo was used to monitor phagocytosis in vivo. To measure clearance of IC-IP6

from the blood, we injected WT and SREBP-1aDF/B6 mice (n = 5 per group) with 10 mmol/kg

4

IC-IP6. At various time-points (0, 1, 4, 8, and 24 h), blood was collected in heparinized tubes,

separated into its constituent parts, and the plasma was stored at -80ºC until further analysis.

Liver and spleen were harvested, washed, and weighed. We fixed half of each tissue

sample in 10% neutral- buffered formalin for histopathological analysis, and homogenized the

other half in saline solution using a tissue lyser (BD Bioscience, Franklin Lakes, NJ, USA).

Tissue lysates were centrifuged, and the supernatant was used for the quantification of total

iron. Total iron and protein concentrations were measured using the AU480 Chemistry System

(Beckman Coulter, Tokyo, Japan) with reagent kits provided by the manufacturer. We

calculated the average iron uptake for each tissue by dividing the mean total iron by the total

protein concentration.

Assessment of iron accumulation. To quantify cellular uptake of IC-IP6, peritoneal

elicited macrophages were cultured in 6-well plates at a density of 5 x 105 cells/ml in medium

that was supplemented with increasing concentrations of IC-IP6 (0 to 0.5 mM) for 24hr at

37ºC in a humidified CO2 incubator. The cells were washed three times with PBS and lysed

with RIPA buffer. Cell lysates were centrifuged, and the supernatants were used for the

quantification of total iron concentration. We calculated the total iron, total protein, and

average iron uptake, as described above. Prussian blue staining was used to assay for iron

accumulation in cells that were grown on chambered cover glasses (Nunc, Fisher Scientific,

Rochester, NY, USA) at a density of 1 x 105 cells/ml in medium for 12 h. Cells were washed

thoroughly in PBS to remove free IC-IP6 and fixed in 10% neutral-buffered formalin for 10

min before staining. The cover glasses were then placed in staining solution (5% potassium

ferrocyanide, 12% HCl) for 1 h, washed three times with distilled water, counterstained with

nuclear fast red, and finally mounted onto slides with mounting medium (Thermoscientific,

Somerset, NJ, USA).

Shotgun lipidomics analysis. BMDMs were isolated from C57B/6J, 1aDF, and

SCAP mKO mice and treated 100 ng/ml LPS for 24 h. Cells were harvested

and then spun down. Cell pellets were homogenized in 0.5 mL 10x diluted PBS in 2.0 ml

cryogenic vials (Corning Life Sciences, Tewksbury, MA) by using a digital sonifier (Branson 450,

Danbury, CT). Protein assay on the homogenates was performed by using a bicinchoninic acid

protein assay kit (Thermo Scientific, Rockford, IL) with bovine serum albumin as standards.

The rest of homogenate was accurately transferred into a disposable glass culture test tube,

5

and a mixture of lipid internal standards was added prior to lipid extraction for quantification

of all reported lipid species. Lipid extraction was performed by using a modified Bligh and

Dyer procedure as described previously(11). Individual lipid extracts were resuspended into a

volume of 100 µL of chloroform/methanol (1:1, v/v) per mg of protein and flushed with

nitrogen, capped, and stored at −20 °C for lipid analysis. For shotgun lipidomics, lipid

extracts were further diluted to a final concentration of ~500 fmol/µL, and the mass

spectrometric analysis was measured usinga QqQ mass spectrometer (Thermo TSQ

Quantiva, San Jose, CA) equipped with an automated nanospray device (TriVersa NanoMate,

Advion Bioscience Ltd., Ithaca, NY) as previously described ( 1 2 ) . Identification and

quantification of lipid molecular species were performed using an automated software program

(13, 14). Data were normalized to per mg of protein.

Salmonella typhimurium infection of BMDM. Salmonella typhimurium (SL1344) ppGpp

deficiency strain was cultured for overnight and then diluted 100-fold into LB broth and grown

for another 4 h at 37 °C. Bacterial inoculums were prepared by pelleting the cells at 10,000

x g for 5 min and pellets were resuspended in Dulbecco's modified Eagle's modified (corning)

plus 10% fetal bovine serum (Hyclone). Monolayers for bacterial invasion were prepared by

seeding 7 × 104 BMDMs into each well of 8-well chamber slides. The macrophages were

cultured in DMEM plus 10% fetal bovine serum (Hyclone) at 37 °C with 5% CO2. The

bacterial inoculums were diluted and added to the BMDM cells at MOI 1:100 for 30 minutes at

37 °C in 5% CO2/95% air. Infected cells were washed three times with phosphate-buffered

saline (pH 7.4), Dulbecco's modified Eagle's medium containing gentamicin (20 ug/ml) was

added, and the mixture was incubated for an additional 30 min. The chamber slides were

sealed with fluorescent mounting medium containing DAPI (Molecular Probes, Eugene, OR,

USA). Images were acquired by confocal laser scanning microscopy (Carl Zeiss, Thornwood,

NY, USA).

Isolation of detergent resistance membrane (DRM) fractions. DRM fractions were

isolated from BMDMs as described previously (15). Briefly, BMDM cells were homogenized in

hypotonic buffer (1 mM HEPES, pH 8.0, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, and

protease inhibitors). Cell lysates were subjected to sequential centrifugation step (2,000 x g

for 5min, 10,000 x g for 15 min, 100,000 x g for 2 h) to yield crude membrane fraction. Cell

pellet is suspended in MES buffer (10 mM MES, 150 mM NaCl, pH 6.5, and protease inhibitors)

6

incubated with 1% Triton X-100 on ice for 30 min, and homogenized by 20 passes through

29-guage needles. Homogenates were mixed with an equal volume of 80% sucrose in MES

buffer. The resulting diluent was placed at the bottom of a continuous sucrose concentration

gradient (30%-5%) in the same buffer. After centrifugation at 39,000 rpm, 4 ℃ for 16 h,

fractions were collected from top to bottom.

For assessment of actin network association with lipid membrane rafts by immunoblotting,

BMDM cells were plated in 150 mm culture dishes (1 ⅹ 107 cells/dish). After 48 h incubation,

cells were treated with 0.5 mM BSA-conjugated OA for 24 h and/or 100 ng/ml LPS for 16 h. Cells

were harvested and isolated DRMs using the same procedures above. Fractions were analyzed

by immunoblotting with anti-moesin and anti-cofilin antibodies.

Immunohistochemistry. Tissue samples were fixed in 10% neutral-buffered formalin and

embedded in paraffin. To confirm the presence of IC-IP6 in the cytoplasm of macrophages,

sections (2.5-um thickness for LNs and 5-um thickness for other tissues) were stained with

Prussian blue, and IHC staining was performed with a rat anti-mouse anti-F4/80 monoclonal

(1:200, clone BM8; eBioscience, San Diego, CA). All images were analyzed using the publicly

available programs Image J (16).

Detection of BODIPY-GM1 by Fluorescent Microscopy. BMDM cells (5 ⅹ104 cells/well) were

seeded overnight onto 8-well chamber glass slides (Lab-Tek II, Nalgene Nunc, Naperville, IL) and

cultured in control medium or media containing 500 µM BSA conjugated OA and/or 100 ng/ml

LPS. After 24 h incubation, the cells were fixed with 4% paraformaldehyde in PBS. The fixed cells

were permeabilized and then incubated in blocking solution (1% BSA and 0.1% Triton-X in PBS)

at room temperature for 1 h followed by incubation with anti-Moesin (ab52490, abcam,

Cambridge, UK) or anti-Cofilin (ab54532, abcam) diluted in blocking solution at 4 °C overnight.

Following incubation with primary antibodies, the cells were incubated with the appropriate Alexa

Fluor-546-conjugated secondary antibodies (Life Technologies, Gaithersburg, MD, USA) for 1 h

at room temperature. After three 3-times washes with DPBS, LD were stained with 500 nM

BODIPY-GM1 (B13950, Life Technologies) for 20 min. The chamber slides were sealed with

fluorescent mounting medium containing DAPI (S36939, Invitrogen Carlsbad, CA). The images

were acquired by confocal laser scanning microscopy (Carl Zeiss, Oberkochen, Germany). Co-

localization coefficient (R) was obtained using the ImageJ plug-in “Colocalization_Finder”.

7

Statistical analysis. All data were expressed as mean ± SD of the number of determinations

carried out in triplicate. Variables were tested for normality and then the different groups were

compared using the Paired Sample t-test, where p < 0.05 was considered as statistically

significant between groups.

Supplemental Figure Legends

Supplementary Figure 1. Phagocytosis level was decreased in SREBP-1aDF BMDMs.

(A) Photomicrographs of a typical experiment in which BMDMs were adhered to human

albumin serum for 30 min prior to addition of opsonized early antigen (EA)-IgG targets. And

then macrophage cell was incubated with sheep blood targets for 30 min at 37°C. And

cells were fixed, stained and photographed. (B) Percentage of phagocytosis was assessed by

counting phagocytic macrophage cells. Arrows indicate the digested targets in BMDMs

isolated from WT and SREBP-1aDF mice. Opsonized IgG antibodies were diluted as

indicated. (C) Phagocytic index was indicated the digested targets which were counted in the

200 cells. (D) Reduced phagocytosis in SCAP mKO BMDMs. Phagocytosis assay using the

IgG-opsonized sheep red blood cell target. LPS (100 ng/ml) was treated for 24 h in BMDMs

of SCAPfl/fl and SCAP mKO mice. Arrows indicate the engulfed targets in BMDMs. (E)

Percentage of phagocytosis was shown by counting phagocytic macrophage cells. (F)

Phagocytic index was indicated. Data are represented as mean ± SEM. **p < 0.01, ***p <

0.001.

Supplementary Figure 2. Reduced phagocytosis of invasion defective Salmonella

typhimurium in SREBP-1aDF/B6 BMDMs. (A) Comparison of the engulfed salmonella in the

WT and SREBP- 1aDF/B6 BMDMs. Salmonella typhimurium with SPI-1 and SPI-2 mutations

were infected in BMDMs for 30 min. Green indicates ppGpp-GFP and Blue is DAPI staining.

(B) Relative fluorescence intensity of ppGpp-GFP in WT and SREBP-1aDF/B6 BMDMs.

Values are expressed as mean +/-SEM. **p < 0.01.

Supplementary Figure 3. Apoptotic cell-mediated phagocytosis assay. Uptake of

apoptotic Jurkats by BMDMs. Jurkat cells were labels with Carboxyfluorescein succinimidyl

ester (CFSE) and treated with 40 uM etoposide for 16 h to induce apoptosis in complete

media. Apoptotic cells were fed to phagocytes. CD11b and CSFE deposition was assessed by

8

flow cytometry. Data were expressed as mean ± SEM. *p < 0.05. Jurkat cells alone or incubated

with normal human serum (NHS) were used as shown in the Figure.

Supplementary Figure 4. Phagocytosis assay in TLR4 KO macrophages. BMDMs were

isolated from C57B6/J and TLR4 KO mice. BMDMs were treated with 100 ng/ml LPS for 16 h

and phagocytosis was measured as described above. Percentage of phagocytosis (A) and

phagocytotic index (B) were measured by counting the phagocytic cells and Ingested targets.

Data are represented as mean ± SEM. **p < 0.01.

Supplementary Figure 5. Representative images and Pearson's coefficient measuring

colocalization of Moesin/BODIPY-Gm1 or Cofilin/BODIPY-Gm1. (A) BMDM, labeled with

BODIPY-Gm1 (green) and moesin antibody (red) were treated with 100 ng/ml of LPS and/or 500

uM OA. Alexa-Fluor-546 was used to conjugate with anti-moesin. BMDMs were analyzed using

confocal laser microscopy. Yellow spots indicate clustering and colocalization of BODIPY-Gm1

and moesin or cofilin. (B) Pearson’s correlation coefficients (r) of moesin were evaluated for the

co-localization regions that could be taken within each cell. (C) immunocytochemistry image with

BODIPY-Gm1 (green) and cofilin antibody (red). (D) Pearson’s correlation coefficients (r) of

cofilin. Data are represented as mean ± SEM. *p<0.05, **p < 0.01, ***p < 0.001 vs WT BMDMs.

Supplemental References

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acid partitioning by activating acetyl coenzyme A carboxylase 2. Mol Cell Biol 29(17):4864-

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19(3):345-346.

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19(3):346-347.

5. Peterson TR, et al. (2011) mTOR complex 1 regulates lipin 1 localization to control the

SREBP pathway. Cell 146(3):408-420.

6. Matsuda M, et al. (2001) SREBP cleavage-activating protein (SCAP) is required for

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increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation.

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cell surface protein involved in the regulation of phagocytic activity. J Immunol

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to activate CR1-mediated phagocytosis by human mononuclear phagocytes. Eur J

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9. Im SS & Osborne TF (2012) Protection from bacterial-toxin-induced apoptosis in

macrophages requires the lipogenic transcription factor sterol regulatory element binding

protein 1a. Mol Cell Biol 32(12):2196-2202.

10. Oh BC, et al. (2006) Ca(2+)-inositol phosphate chelation mediates the substrate specificity

of beta-propeller phytase. Biochemistry 45(31):9531-9539.

11. Bligh EG & Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J

Biochem Physiol 37(8):911-917.

12. Han X, Yang K, & Gross RW (2008) Microfluidics-based electrospray ionization enhances

the intrasource separation of lipid classes and extends identification of individual

molecular species through multi-dimensional mass spectrometry: development of an

automated high-throughput platform for shotgun lipidomics. Rapid Commun Mass

Spectrom 22(13):2115-2124.

13. Yang K, Cheng H, Gross RW, & Han X (2009) Automated lipid identification and

quantification by multidimensional mass spectrometry-based shotgun lipidomics. Anal

Chem 81(11):4356-4368.

14. Wang M, Wang C, Han RH, & Han X (2016) Novel advances in shotgun lipidomics for

biology and medicine. Prog Lipid Res 61:83-108.

15. Wei X, et al. (2016) Fatty acid synthesis configures the plasma membrane for inflammation

in diabetes. Nature 539(7628):294-298.

16. Schneider CA, Rasband WS, & Eliceiri KW (2012) NIH Image to ImageJ: 25 years of

image analysis. Nat Methods 9(7):671-675.

A

B C% Phagocytosis

1:160 1:320 1:6400

20

40

60

80WT1aDF

% P

hago

cyto

sis

Phagocytosis index

1:160 1:320 1:6400

50

100

150

200

250WT1aDF

Phag

ocyt

osis

inde

x

WT

1aDF

1:160 1:320 1:640

**

*****

***

****

D E

F

SCAPfl/fl

SCAP mKO

Mock LPS% Phagocytosis

Mock LPS0

10

20

30

40

SCAP fl/fl

SCAP mKO

% P

hago

cyto

sis

Phagocytic index

Mock LPS0

20

40

60

Pha

gocy

tic in

dex

**

**

***

***

Lee et al. 2018 Supplemental Figure 1.

DAPI GFP Merge

WT

1aDF

Infection Salmonella with SPI-1 and SPI-2 mutant strain (30min)

BMDM cellGreen : ΔPPGPP-GFPBlue : DAPI

A

Salmonella infection (ppGpp)

WT 1aDF0.0

0.5

1.0

1.5

Rel

ativ

e flu

ores

cenc

e in

tens

ity

B

*

Lee et al. 2018 Supplemental Figure 2.

A

B

C Apoptotic cell phagocytosis

Jurkats Jurkats+NHS0

10

20

30

40

*WT1aDF

% P

hago

cyto

sis

Lee et al. 2018 Supplemental Figure 3.

Lee et al. 2018 Supplemental Figure 4.

Phagocytosis assay in TLR4 KO BMDMs

% Phagocytosis

Mock LPS0

20

40

60TLR4+/+ TLR4-/-

% P

hogo

cyto

sis

Phagocytic index

Mock LPS0

50

100

150TLR4+/+ TLR4-/-

Phag

ocyt

ic in

dex

A B

Red : MoesinGreen : BODIPY-GM1Blue : DAPI

BSA

LPS OA LPS+OA

WT

1aDF

40xCo-localization coefficient (R)

between Moesin and BODIPY-GM1

BSA LPS OA LPS+OA0.0

0.2

0.4

0.6WT1aDF

****

Co-

loca

lizat

ion

coef

ficie

nt (R

)

*

40x BSA LPS OA LPS+OA

WT

1aDF

Co-localization coefficient (R)between Cofilin and BODIPY-GM1

BSA LPS OA LPS+OA0.0

0.1

0.2

0.3

0.4WT1aDF

Co-

loca

lizat

ion

coef

ficie

nt (R

)

*

***

**

Red : CofilinGreen : BODIPY-GM1Blue : DAPI

A B

C D

Lee et al. 2018 Supplemental Figure 5.