Yu et al: Rosuvastatin reduces aortic sinus and coronary artery atherosclerosis in SR-B1/apoE dko mice. Supplementary Tale i: Primers used for RT-PCR. References are provided under Source, except for primers designed using PuMed s Primer-BLAST. Target Primer Sequence Source Arginase-1 Forward 5 -GCTCAGGTGAATCGGCCTTTT-3 1 5 -TGGCTTGCGAGACGTAGAC-3 CD36 Forward 5 - TTTCCTCTGACATTTGCAGGTCTA-3 5 -AAAGGCATTGGCTGGAAGAA-3 Fizz-1 Forward 5 - CACCTCTTCACTCGAGGGACAGTTG-3 1 5 - GGTCCCAGTGCATATGGATGAGACC-3 GAPDH Forward 5 -ACCACAGTCCATGCCATCAC-3 5 -TCCACCACCCTGTTGCTGTA-3 IL-1β Forward 5 - AGGCAGGCAGTATCACTCATTGT-3 2 5 - GGAAGGTCCACGGGAAAGA-3 IL-6 Forward 5 -TAGTCCTTCCTACCCCAATTTCC-3 1 5 - TTGGTCCTTAGCCACTCCTCC-3 LDLR Forward 5 -TCCAATCAATTCAGCTGTGG-3 Mannose receptor 5 -GAGCCATCTAGGCAATCTCG-3 Forward 5 - AAACACAGACTGACCCTTCCC-3 3 5 - GTTAGTGTACCGCACCCTCC-3 MCP-1 Forward 5 - TTCCTCCACCACCATGCAG-3 4 5 - CCAGCCGGCAACTGTGA-3 PCSK9 Forward 5 -TTGCAGCAGCTGGGAACTT-3 5 5 -CCGACTGTGATGACCTCTGGA-3 SR-A Forward 5 - CTTTACCAGCAATGACAAAAGAGA-3 5 -ATTTCACGGATTCTGAACTGC-3 1
Yu et al: Rosuvastatin reduces aortic sinus and coronary artery atherosclerosis in SR-B1/apoE dko mice. Supplementary Tale ii: Antiodies used in this study. IF indicates immunofluorescence, Flow indicates flow cytometry. Antiody Supplier Catalogue # Use Primary Goat anti-human apoa1 Midland Bioproducts 71107 Immunolot Goat anti-human apob Midland Bioproducts 71301 Immunolot Rait anti-apob48 receptor Acam A125128 Immunolot FITC rat anti-mouse CD3 BD Pharmingen 555274 Flow APC rat anti-mouse CD11 (M1/70) Invitrogen RM2805 Flow FITC rat anti-mouse CD11 (M1/70) BD Pharmingen 553310 Flow Rait anti-mouse CD36 Santa Cruz Biotech. SC9154 IF APC-anti-mouse CD36 BioLegend 102611 Flow Rat anti-mouse CD41 (MWReg30) BD Pharmingen 553847 IF PerCP-Cy5.5 anti-hu/mo CD45R/B220 ebioscience 45-0452-80 Flow E06 IgM monoclonal antiody Avanti Polar Lipids 330001 IF Rait anti-ε-cop Novus Biologicals NBP2-38512 Immunolot F4/80 monoclonal antiody Acam a6640 IF/Flow FITC anti-gp1β Emfret Analytics M050-1 Flow PE-JON/A Emfret Analytics D200 Flow (anti- GPII/IIIa) Goat anti-mouse LDLR R&D Systems AF2255 Immunolot Rait anti-lrp-1 Acam a92544 Immunolot Mouse anti-mouse Na + /K + ATPase α-1 Upstate Technology 05-369 Immunolot FITC anti-p-selectin Emfret Analytics D200 Flow Secondary Alexa-488 goat anti-mouse IgM Invitrogen A21042 IF Alexa-488 goat anti-rat Invitrogen A11006 IF Alexa-594 goat anti-rait Invitrogen A11012 IF/Flow 2
Yu et al: Rosuvastatin reduces aortic sinus and coronary artery atherosclerosis in SR-B1/apoE dko mice. HRP donkey anti-mouse IgG Jackson Immunoresearch 715-035-150 Immunolot HRP donkey anti-rait IgG Jackson Immunoresearch 715-035-152 Immunolot HRP rait anti-goat IgG Jackson Immunoresearch 305-035-003 Immunolot 3
Yu et al: Rosuvastatin reduces aortic sinus and coronary artery atherosclerosis in SR-B1/apoE dko mice. Legends to Supplementary Figures Supplementary Figure i: LRP-1 and apob48r levels in liver total memranes of control and rosuvastatin treated SR-B1 -/- /apoe -/- mice. Representative immunolots (A,C) and quantification (B,D) of LRP-1 (A,B) or apob48r (C,D) levels in liver total memranes from control saline- or rosuvastatin-treated SR-B1 -/- /apoe -/- mice (n=5, 5). ε-cop was used to control for equal loading. NS indicates not statistically significantly different y Mann-Whitney rank sum test. Supplementary Figure ii: Scatter plots of atherosclerosis in aortic sinuses and coronary arteries from rosuvastatin and control treated SR-B1 -/- apoe -/- mice. Scatter plots are shown for the atherosclerosis data presented in figure 2C for the aortic sinus (panel A) and figure 2I for coronary arteries (panel B). Red symols correspond to female and lue to male mice. Horizontal ars and whiskers correspond to means±sem of pooled data. Values for control females and control males were not statistically significantly different (P=0.76 for panel A, and P=0.17 for panel B). Values for control versus rosuvastatin treated mice were statistically significantly different for females (P=0.029 for panel A and P=0.0007 for panel B) and when oth males and females were pooled (P=0.023 for panel A and P<0.0001 for panel B). Mann-Whitney rank sum test was used for statistical analysis. Supplementary Figure iii: Flow cytometry analysis of platelets. Whole heparinized lood was collected y carotid artery cannulation, laeled with antiodies and fixed as descried in the methods section. Flow cytometry was performed using a BD FACSCaliur. Forward and side scatter was used to identify platelets (gated as shown). Shown are forward and side scatter and gating for platelets for representative samples from a apoe -/- sko control mouse (left panel) and SR-B1 -/- apoe -/- dko mice treated with saline (control-middle panel) or rosuvastatin (right panel). Supplementary Figure iv: Platelet CD36 levels detected y flow cytometry. Whole lood (26 μl) was incuated with 2 μl FITC-conjugated anti-gp1β (to identify platelets) and 1 μl APC-conjugated anti-cd36 for 15 min, and then mixed with 0.4 ml PBS containing 1 % formaldehyde (freshly prepared from paraformaldehyde), prior to flow cytometry analysis using a BDFACSCaliur flow cytometer. Platelets were identified y gating for FITC (GP1β+) and the median APC fluorescence (CD36) of the histogram was determined for each sample. C indicates control, saline-treated. R indicates rosuvastatin-treated. sko are apoe -/- and dko are SR-B1 -/- /apoe -/- mice. Data are averages ± S.E.M. of N=4 (sko) and N=5 (C and R, dko) mice. Data were analyzed y the Kruskall-Wallis one-way ANOVA and found not to e statistically significantly different (P>0.4). Supplementary Figure v: Flow cytometry analysis of leukocytes from control saline- or rosuvastatin treated SR-B1 -/- /apoe -/- mice. Blood from control saline- and rosuvastatin-treated SR- B1 -/- apoe -/- dko mice was treated to lyse erythrocytes, washed and laeled as descried in the Methods section with A) FITC-laeled rat anti-mouse CD3 (for T-lymphocytes), B) PerCP-Cy5.5-laeled antihu/mo CD45R/B220 (for B-lymphocytes) and C) APC-laeled anti-mouse CD11 (for myeloid cells). The proportion of cells positive for each of the three markers was determined y flow cytometry using a BD FACSCalier instrument. Shown are the means from N=6 control saline- and N=5 rosuvastatin treated SR-B1 -/- apoe -/- dko mice. Data was analyzed y the Mann-Whitney rank sum test. Rosuvastatin treatment did not significantly affect levels of circulating CD3 +, B220 + or CD11 + leukocytes (P>0.05). 4
Yu et al: Rosuvastatin reduces aortic sinus and coronary artery atherosclerosis in SR-B1/apoE dko mice. Supplementary Figure vi: Rosuvastatin treatment of one marrow derived macrophages from SR-B1 -/- /apoe -/- mice did not affect polarization towards M1 or M2 phenotypes. Bone marrow was prepared from femurs and tiias of N=3 SR-B1 -/- apoe -/- dko mice. Cells were plated overnight in DMEM with 10 % FBS and cells in suspension were recovered, washed and cultured for 7 days in DMEM containing 10 % FBS and 20 % L-cell conditioned medium to allow for differentiation to macrophages. Macrophages were treated with or without 10 μm rosuvastatin, and 10 ng/ml of either LPS (for M1 polarization, A-C) or IL-4 (for M2 polarization, D-F) as indicated. After 6 hrs, cells were lysed and total RNA was extracted, purified and quantified and analyzed y quantitative real time PCR as descried in the Methods section. LPS treated samples were analyzed for A) IL-1β, B) IL-6 and C) MCP-1 (top) and IL-4 treated samples were analyzed for D) arginase 1 (ARG-1), E) mannose receptor and F) Fizz-1 (ottom). GAPDH was analyzed for all samples. Primers are shown in supplementary tale 1. Levels of target product were normalized to GAPDH and are expressed as fold change relative to control cells not treated with either LPS or IL-4. Values are means ± standard error of the mean of N=3 one marrow derived macrophage preparations. Data was analyzed y two-way ANOVA using a Holme-Sidak post test. Different lower case letters indicate statistically significantly different values (P<0.05). Supplementary Figure vii: Rosuvastatin treatment protects mouse peritoneal macrophages from apoptosis. Thioglycollate-elicited peritoneal macrophages from wild type mice (n=3) were cultured on chamer slides in DMEM containing 3 % neworn calf lipoprotein deficient serum and treated for 24 hrs with either no additions, or with 1 μm thapsigargin without or with 20 μm rosuvastatin. Apoptosis was detected y FITC-Annexin V staining (green fluorescence-see arrows). DAPI was used to counter stain nuclei. A-C) Show representative images. Arrows indicate Annexin V + cells. D) The proportion of Annexin V + cells is plotted as averages ± S.E.M. of cells from N=3 mice. Data was analyzed y oneway ANOVA with the Tukey s multiple comparisons test. *** : P=0.0003; **: P=0.005. 5
Yu et al: Rosuvastatin reduces aortic sinus and coronary artery atherosclerosis in SR-B1/apoE dko mice. Supplementary References 1. Sanson M, Distel E, Fisher EA. Hdl induces the expression of the m2 macrophage markers arginase 1 and fizz-1 in a stat6-dependent process. PloS one. 2013;8:e74676 2. Li AC, Binder CJ, Gutierrez A, Brown KK, Plotkin CR, Pattison JW, Valledor AF, Davis RA, Willson TM, Witztum JL, Palinski W, Glass CK. Differential inhiition of macrophage foamcell formation and atherosclerosis in mice y pparalpha, eta/delta, and gamma. The Journal of clinical investigation. 2004;114:1564-1576 3. Stein M, Keshav S, Harris N, Gordon S. Interleukin 4 potently enhances murine macrophage mannose receptor activity: A marker of alternative immunologic macrophage activation. The Journal of experimental medicine. 1992;176:287-292 4. Trogan E, Choudhury RP, Dansky HM, Rong JX, Breslow JL, Fisher EA. Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein e-deficient mice. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:2234-2239 5. Ai D, Chen C, Han S, Ganda A, Murphy AJ, Haeusler R, Thorp E, Accili D, Horton JD, Tall AR. Regulation of hepatic ldl receptors y mtorc1 and pcsk9 in mice. The Journal of clinical investigation. 2012;122:1262-1270 6
Supplementary figure i LRP-1 and apob48r levels in livers of rosuvastatin and control treated mice A Rosuvastatin - + B NS LRP1 ε - COP 85 kda 35 kda C Rosuvastatin - + D Control Rosuvastatin NS ApoB48R ε - COP 250 kda 35 kda Control Rosuvastatin 7
Supplementary figure ii Scatter plots of atherosclerosis in aortic sinuses and coronary arteries from rosuvastatin and control treated SR-B1 -/- apoe -/- mice A: Aortic Sinus Atherosclerosis B: Coronary Artery Atherosclerosis 8
Supplementary figure iii Flow cytometry analysis of platelets apoe -/- SR-B1 -/- apoe -/- Control Control SR-B1 -/- apoe -/- Rosuvastatin 9
Supplementary figure iv Platelet CD36 levels Anti-CD36 (m edian fluorescence aritrary units) 20 15 10 5 0 ro l NS C C R ro l tin sko dko 10
Supplementary figure v Flow cytometry analysis of leukocytes A B C 11
Supplementary figure vi Polarization of one marrow derived macrophages Macrophage M1 Polarization A B C a a Control Rosuvastatin Control Rosuvastatin No LPS LPS a a Control Rosuvastatin Control Rosuvastatin No LPS LPS a a Control Rosuvastatin Control Rosuvastatin No LPS LPS Macrophage M2 Polarization D E F a a a a a a Control Rosuvastatin Control Rosuvastatin No IL-4 IL-4 Control Rosuvastatin Control Rosuvastatin No IL-4 IL-4 Control Rosuvastatin Control Rosuvastatin No IL-4 IL-4 12
Supplementary figure vii Protection of macrophages against apoptosis B -Thapsigargin C +Thapsigargin -Rosuvastatin -Rosuvastatin A +Rosuvastatin B C c e lls + A n n e x in V (p r o p o r tio n o f to ta l) 0.4 D *** ** 0.3 0.2 0.1 0.0 20 μm Rosuvastatin 1 μm Thapsigargin 13 - - + +