McAlpine PERK-GSK3 regulates foam cell formation. Supplemental Material. Supplementary Table I. Sequences of real time PCR primers.
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1 Mclpine PERK-GSK3 regulates foam cell formation Supplemental Material Supplementary Table I. Sequences of real time PCR primers. Primer Name Primer Sequences (5-3 ) Product Size (bp) GRP78 (human) Fwd: CG GG GG GG C G GG 86 Rev: CC CTT G CGG C G CT GRP78 (mouse) Fwd: CC TGG GTG GGG C CT TT 1 Rev: TCT TC T TTG GCC CG GT GRP9 Fwd: TGG G GG GTT CC G TG 19 Rev: GTT GCC G CC TCC GT CT Calreticulin Fwd: TG G GG CCC CGT TCT TCC T 115 Rev: G CC CG GCC TG GT TTC TC T PDI Fwd: G CTC GC GC CC 159 Rev: CC TT TT CC GGC CC CT TF Fwd: CTC CGG GC G TTG GT GTT 153 Rev: GGC TGC TT GTC TCC TGG C CHOP (human) Fwd: TGC CTT TCT CTT CGG C CT Rev: TGT GC CTC TGC TGG TTC TG CHOP (mouse) Fwd: GTC CCT GC TTG GCT GC G 1 Rev: TGG G GCG GG GCT TTG FS Fwd: G GC CTG TCT GG TTT GT GC 15 Rev: TGG CTT CT GG TG CTT CC SREP Fwd: C GT GC C GTC TGG CG Rev: GCT TC GC CC TGT TCT CCT G SREP1c Fwd: CG CT CG G CT GCT TC 138 Rev: GG GG CTT C GG GG GC HMGCo Fwd: CTG TC TTC CG CC GG TTG 167 Rev: GTC CC GG C TGT G TGG GLUD Fwd: CCG TT CG CC TG TGT GG 169 Rev: TGG TG CC TCC TTG TG TCT Citrate Synthase Fwd: TGC TTC CTC CC G TTT G 131 Rev: CC CC TC TC TG TCC C G βctin (human) Fwd: CC GG CGC GGC TC G 5 Rev: CTT T GTG CG CC GT TTC C βctin (mouse) Fwd: GGC CC C CCT TCT C TG Rev: GGG GTG TTG G GTC TC C 9 S1
2 GSK3α/β activity (percent of control) OD (57nm).7.6 y =.31x -. R² = μg cholesterol Supplementary Figure I. Validation of cholesterol assay (Sigma ldrich catmk3). Colorimetric assay conducted according to manufacturers instructions on cholesterol standard. 1 Col 1 Col 3 1 GSK3α GSK3β β catenin whole cell lysates - + CT μmol/L CT991 Supplementary Figure II. CT991 inhibits GSK3α and GSK3β kinase activity. GSK3α and GSK3β were immunoprecipitated from whole cell Thp-1 macrophage lysates. Kinase activity was determined in the presence or absence of.5μmol/l CT991 (). Whole cell lysates from Thp-1 macrophages treated in the presence or absence of μmol/l CT991 were resolved by SDS page and probed for β catenin to demonstrate GSK3α/β inhibition (). n=3 p<.5 relative to cells of the same treatment without GSK3α/β inhibition S1
3 lipid accumulation/cell % Cell Viability % Cell Viability CT991 UT Thaps GLN P UT 1μM Dox 1.5μM Dox μm Dox C μm Dox D μm Dox Supplementary Figure III. poptosis does not induce foam cell formation. () Thp1 derived macrophages were exposed to the ER stress-inducing agents thapsigargin (Thaps, 1μmol/L), glucosamine (GLN, 5mmol/L) or palmitic acid (P, μmol/l) with or without pretreatment with CT991. Cell viability was determined after 18 hours. () Thp1 derived macrophages were exposed to, 1, 1.5 or μm Doxorubicin (Dox) for 6 hours and stained with Oil Red O, arrows indicate positive staining. (C) Quantification of Oil Red O staining. (D) Dox exposure induced apoptosis and reduced viability of Thp1 macrophages n=3, p<.5 relative to untreated cells. S
4 lipid accumulation/cell GRP78/ CHOP/ sirn sirn sirn sirn sirn UT Thaps GLN P GSK3α - GSK3β - GRP78 CHOP C 8 sirn GSK3α/β sirn D 5 sirn GSK3α/β sirn E UT Thaps GLN P UT Thaps GLN P UT Thaps GLN P sirn GSK3α/β sirn F 5 sirn GSK3α/β sirn 3 1 UT Thaps GLN P S3
5 Supplementary Figure IV. Knockdown of GSK3α/β by sirn attenuates ER stress-induced CHOP expression and lipid accumulation. Thp1 derived macrophages were exposed to 5nmol/L scramble or GSK3α/β sirn. Knockdown of GSK3α/β protein levels was confirmed by SDS PGE(). Cells were subsequently treated with the ER stress-inducing agents thapsigargin (Thaps, 1μmol/L), glucosamine (GLN, 5mmol/L) or palmitic acid (P, μmol/l) for 18 hours. Whole cell lysates were resolved by SDS PGE and probed with antibodies against GRP78 and CHOP (). Immunoblots were quantified by densitometry (C and D). Cells were stained with Oil Red O and DPI and representative images are shown (E) and quantified (F). n= p<.5 relative to scramble sirn, untreated cells, p<.5 relative to cells of the same treatment without GSK3α/β sirn UT Thaps - + PERK inh CHOP Supplementary Figure V. PERK inhibitor (GSK61) attenuates PERK signaling. Whole well lysates from Thp-1 derived macrophages treated with 1μM thapsigargin (Thaps) in the presence or absence of 3μmol/L GSK61 (PERK Inhibitor) were resolved by SDS-PGE and probed for CHOP to demonstrate PERK inhibition. DPI Rat IgG Mouse IgG Rabbit IgG DPI Mac3 KDEL CHOP Supplementary Figure VI. IgG control antibodies. ortic sections were exposed to () pre-immune rat or mouse IgG antibodies to control for non specific binding of antibodies against Mac3 and KDEL respectively and () rabbit IgG antibodies to control for non specific binding of antibodies against CHOP. S
6 mrn/βctin mrn/βctin Glutamate Dehydrogenase ns CT991 UT Thaps GLN P Citrate Synthase Supplementary Figure VII. ER stress and CT991 do not impact the expression of glutamate dehydrogenase or citrate synthase. Thp1 derived macrophages were exposed to the ER stress-inducing agents thapsigargin (Thaps, 1μmol/L), glucosamine (GLN, 5mmol/L) or palmitic acid (P, μmol/l) for 18 hours with or without pretreatment with CT991. The mrn expression of () Glutamate Dehydrogenase and () Citrate Synthase was determined by real time PCR. n=3, ns = not significant. ns CT991 UT Thaps GLN P S5
7 μg cholesterol / mg protein μg cholesterol / mg protein Free Cholesterol UT - CT - CT - CT GSK3α/β inhibitor Thaps GLN P Cholesteryl Esters UT - CT - CT - CT Thaps GLN P GSK3α/β inhibitor Supplementary Figure VIII. ER stress-induced cholesterol accumulation is attenuated by different GSK3α/β inhibitors. ER stress was induced in Thp-1 macrophages using 1μmol/L thapsigargin (Thaps), 5mmol/L glucosamine (GLN) or μmol/l palmitic acid (P). GSK3α/β was inhibited by treating cells with μmol/l CT991 (CT), or 5μmol/L GSK3 inhibitor II (Inh ), or mmol/l valproate (VP). Free () and esterified () cholesterol levels were determined, as indicated. n=3-, p<.5 relative to untreated cells, p<.5 relative to cells of the same treatment without GSK3α/β inhibition. S6
8 GSK3β/ μg cholesterol / mg protein μg cholesterol / mg protein Free Cholesterol Cholesteryl Esters CT991 UT Thaps GLN P Supplementary Figure IX. GSK3α/β inhibition attenuates free cholesterol synthesis in macrophages cultured in lipoprotein-deficient media. ER stress was induced in Thp-1 macrophages cultured in lipoprotein free media using 1μmol/L thapsigargin (Thaps), 5mmol/L glucosamine (GLN) or μmol/l palmitic acid (P). GSK3α/β was inhibited by treating cells with μmol/l CT991. Free () and esterified () cholesterol levels were determined. ). n=3-, p<.5 relative to untreated cells, p<.5 relative to cells of the same treatment without CT991. GSK3β d-cmv- Null d-s9- GSK3β right field CT991 UT Thaps GLN P d-cmv- Null GFP d-s9- GSK3β d-null d-gfp Supplementary Figure X. denovirus directed overexpression of GSK3β. Primary mouse macrophages were infected with control adenovirus (d-null) or adenovirus encoding constitutively active GSK3β (d-s9-gsk3β) at 1 MOI. () Protein lysates were resolved by SDS-PGE immunostained for GSK3β and as indicated and () quantified. Similarly, primary mouse macrophages were infected with d-null or adenovirus encoding GFP (d-gfp) at 1 MOI. right field and florescent images were captured and the percentage of cell expressing GFP was quantified (C). Quantification indicates 65.7±3.6% of cells are infected with the adenovirus. n=3 p<.5 relative to d-null S7
SUPPLEMENTAL FIGURE 1
SUPPLEMENTL FIGURE 1 C Supplemental Figure 1. pproach for removal of snorns from Rpl13a gene. () Wild type Rpl13a exonintron structure is shown, with exo in black and intronic snorns in red rectangles.
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Supplementary Materials
Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi
c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
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Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
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Supplementary Document
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CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'
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