Supplementary Figures: Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or 1 mg/kg of D4F (D4F group) per day by intraperitoneal injection during the final 6 weeks. Male C57BL/6J mice were maintained on normal chow diet as a control group. (A) Body weights of mice at 0, 4 and 8 weeks. (B) Levels of serum total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) in mice at the end of experiment. Non-HDL-C was calculated as TC minus HDL-C. Data are presented as the mean ± SEM of at least eight independent experiments. **P<0.01 versus control group. Supplementary Figure S II: D4F reduces the upregulation of CHOP and CD36 in atherosclerotic lesions of apoe -/- mice. Mice were treated as described in Supplementary Figure S I. (A) Representative immunostained aortic sinus sections using specific antibodies against MOMA-2, CHOP and CD36. Scale bar = 50 μm. (B) Densitometric quantification of MOMA-2, CHOP and CD36 positive cells per field of view. Data are presented as the mean ± SEM of at least three independent experiments. #P<0.05 versus model group. Supplementary Figure S III: D4F attenuates TM-induced apoptosis in RAW264.7 cells. Cells were pretreated with D4F (50 mg/l) for 1 h, followed by incubation with TM (4 mg/l) for 24 h, and then cell apoptosis was detected using flow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). Data are expressed as the mean ± SEM of six independent experiments. *P<0.05, **P<0.01 versus control group; &P<0.05 versus TM group. Supplementary Figure S IV: The inhibitory effects of D4F on ox-ldl-induced macrophage 1
apoptosis, caspase-3 activation and CHOP upregulatin were promoted by PBA and blocked by TM. RAW264.7 cells were pretreated with D4F (50 mg/l) for 1 h, washed and followed by treatment with PBA (5 mmol/l) or TM (4 mg/l) for 1 h, and then incubated with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected by TUNEL assay. Scale bar =20 µm. (B) Caspase-3 activity was determined by colorimetric assay. (C) Western blot analysis of CHOP. Data are expressed as the mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01 versus control group. Supplementary Figure S V: D4F inhibits TM-induced mrna upregulation of CHOP and GRP78 in RAW264.7 cells. Cells were pretreated with D4F (50 mg/l) or sd4f (50 mg/l) in the presence of TM (4 mg/l) treatment for 24 h, and then mrna levels of CHOP and GRP78 were evaluated by quantitative real-time PCR. Data are expressed as the mean ± SEM of at least four independent experiments. *P<0.05, **P<0.01 versus control group; &P<0.05 versus TM group. Supplementary Figure S VI: D4F inhibits ox-ldl-induced apoptosis and CHOP upregulation in mouse peritoneal macrophages. Peritoneal macrophages from C57BL/6J mice were harvested with PBS 3 days after intraperitoneal injection with 1 ml of 4% thioglycollate and maintained in DMEM with 10% FBS. Cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected using flow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). (B) Western blot analysis of CHOP. Data are expressed as the mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01 versus control group; #P<0.05 2
versus ox-ldl group. Supplementary Figure S VII: D4F inhibits ox-ldl-induced apoptosis and CHOP upregulation in human THP-1-derived macrophages. THP-1 cells were treated with Phorbol 12-myristate 13-acetate (PMA, 100 nm) for 3 days to induce a macrophage phenotype of differentiation. Cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected by TUNEL assay. Scale bar =20 µm. (B) Western blot analysis of CHOP. Data are expressed as the mean ± SEM of at least three independent experiments. **P<0.01 versus control group; #P<0.05, ##P<0.01 versus ox-ldl group. Supplementary Figure S VIII: CD36 silencing does not promote significantly the inhibitory effects of D4F on ox-ldl-induced apoptosis and CHOP upregulation. RAW264.7 cells were transfected with a sirna against CD36 or a negative control sirna for 48 h, and then the cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected using flow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). (B) Caspase-3 activity was determined by colorimetric assay. (C) Western blot analysis of CHOP. (D) The silencing of CD36 was validated by Western blot. Data are expressed as mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01 versus control group transfected with con-sirna; #P<0.05, ##P<0.01 versus ox-ldl group transfected with con-sirna. NS indicates no significant difference. The sirna sequences of CD36 sirna are 5' -GAAUUU GUCCUAUUGGCCAdTdT-3' (Forward) and 5' -UGGCCAAUAGGACAAAUUCdTdT-3' (Reverse). 3
Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or 1 mg/kg of D4F (D4F group) per day by intraperitoneal injection during the final 6 weeks. Male C57BL/6J mice were maintained on normal chow diet as a control group. (A) Body weights of mice at 0, 4 and 8 weeks. (B) Levels of serum total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) in mice at the end of experiment. Non-HDL-C was calculated as TC minus HDL-C. Data are presented as the mean ± SEM of at least eight independent experiments. **P<0.01 versus control group. Supplementary Figure S II: D4F reduces the upregulation of CHOP and CD36 in atherosclerotic lesions of apoe -/- mice. Mice were treated as described in Supplementary Figure S I. (A) Representative immunostained aortic sinus sections using specific antibodies against MOMA-2, CHOP and CD36. Scale bar = 50 μm. (B) Densitometric quantification of MOMA-2, CHOP and CD36 positive cells per field of view. Data are presented as the mean ± SEM of at least three independent experiments. #P<0.05 versus model group. 4
Supplementary Figure S III: D4F attenuates TM-induced apoptosis in RAW264.7 cells. Cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with TM (4 mg/l) for 24 h. Cell apoptosis was detected using flow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). Data are expressed as the mean ± SEM of six independent experiments. *P<0.05, **P<0.01 versus control group; &P<0.05 versus TM group. 5
Supplementary Figure S IV: The inhibitory effects of D4F on ox-ldl-induced macrophage apoptosis, caspase-3 activation and CHOP upregulatin were promoted by PBA and blocked by TM. RAW264.7 cells were pretreated with D4F (50 mg/l) for 1 h, washed and followed by treatment with PBA (5 mmol/l) or TM (4 mg/l) for 1 h, and then incubated with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected by TUNEL assay. Scale bar =20 µm. (B) Caspase-3 activity was determined by colorimetric assay. (C) Western blot analysis of CHOP. Data are expressed as the mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01 versus control group. 6
Supplementary Figure S V: D4F inhibits TM-induced mrna upregulation of CHOP and GRP78 in RAW264.7 cells. Cells were pretreated with D4F (50 mg/l) or sd4f (50 mg/l) in the presence of TM (4 mg/l) treatment for 24 h, and then mrna levels of CHOP and GRP78 were evaluated by quantitative real-time PCR. Data are expressed as the mean ± SEM of at least four independent experiments. *P<0.05, **P<0.01 versus control group; &P<0.05 versus TM group. 7
Supplementary Figure S VI: D4F inhibits ox-ldl-induced apoptosis and CHOP upregulation in mouse peritoneal macrophages. Peritoneal macrophages from C57BL/6J mice were harvested with PBS 3 days after intraperitoneal injection with 1 ml of 4% thioglycollate and maintained in DMEM with 10% FBS. Cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected using flow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). (B) Western blot analysis of CHOP. Data are expressed as the mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01 versus control group; #P<0.05 versus ox-ldl group. 8
Supplementary Figure S VII: D4F inhibits ox-ldl-induced apoptosis and CHOP upregulation in human THP-1-derived macrophages. THP-1 cells were treated with Phorbol 12-myristate 13-acetate (PMA, 100 nm) for 3 days to induce a macrophage phenotype of differentiation. Cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected by TUNEL assay. Scale bar =20 µm. (B) Western blot analysis of CHOP. Data are expressed as the mean ± SEM of at least three independent experiments. **P<0.01 versus control group; #P<0.05, ##P<0.01 versus ox-ldl group. 9
Supplementary Figure S VIII: CD36 silencing does not promote significantly the inhibitory effects of D4F on ox-ldl-induced apoptosis and CHOP upregulation. RAW264.7 cells were transfected with a sirna against CD36 or a negative control sirna for 48 h, and then the cells were pretreated with D4F (50 mg/l) for 1 h, washed and then followed by incubation with ox-ldl (100 mg/l) for 24 h. (A) Cell apoptosis was detected using flow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). (B) Caspase-3 activity was determined by colorimetric assay. (C) Western blot analysis of CHOP. (D) The silencing of CD36 was validated by Western blot. Data are expressed as mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01 versus control group transfected with con-sirna; #P<0.05, ##P<0.01 versus ox-ldl group transfected with con-sirna. NS indicates no significant difference. The sirna sequences of CD36 sirna are 5' -GAAUUU GUCCUAUUGGCCAdTdT-3' (Forward) and 5' -UGGCCAAUAGGACAAAUUCdTdT-3' (Reverse). 10