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Additional Supporting Information may be found in the online version of this article.

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HEP_23935_sm_suppFig-S1.tif2816KSupplemental Figure 1 Active caspase 3 representative immunostaining of liver tissue sections from WT and CD154KO mice (n=2 mice in each group). Bottom inserts depict a tissue section from CCl4-treated CD154KO mouse, at two different magnifications, as a positive control.
HEP_23935_sm_suppFig-S2.tif3127KSupplemental Figure 2 (A): Active caspase 3 representative immunostaining of liver tissue sections from WT and CD154KO mice 24 hours after vehicle injection and 8 and 24 hours after TM injection. Bottom inserts depict a tissue section from CCl4-treated CD154KO mouse at two different magnifications, as a positive control. (B): Serum concentrations of ASAT and ALAT in WT and CD154KO mice 24 hours after vehicle injection and 8 and 24 hours after TM injection (mean ± SD, n=4 mice in each group; * p<0.05). (C): Immunoblots (representative of 3 performed) of CHOP, Phospho-JNK (P-JNK) and total JNK, from livers of WT and CD154KO mice 24 hours after vehicle injection and 8 and 24 hours after TM injection (CNX, calnexin).
HEP_23935_sm_suppFig-S3.tif1528KSupplemental Figure 3: Expression of CD40 on primary cultures of human and murine hepatocytes and on hepatocyte-derived cell lines and in mouse liver (A): Flow cytometry analysis of CD40 expression on hepatocytes in primary culture and on hepatocyte-derived cell lines. X-axis represents the CD40 fluorescence and the Y-axis the number of events. Gray histogram shows isotype controls, white histograms shows CD40 expression (representative of 3 performed). (B): CD40 immuno-electron microscopy of representative liver tissue sections in WT mice (n=3) at low magnification (top panels) and high magnification (bottom panels). Bottom panels refer to high magnifications of the respective inserts depicted in top panels (C): CD40 and CNX immunoblots from WT and CD154KO liver lysates from mice fed for 3 weeks on sham diet or on olive oil-rich diet (Olive oil diet).
HEP_23935_sm_suppFig-S4.tif172KSupplemental Figure 4: eIF2α phosphorylation in oleic acid and tunicamycin-treated HepG2 cells. Immunoblots of Phospho-eIF2α (P-eIF2α and total eIF2α. from HepG2 cell lysates following incubation with (A) TM (2.5 μM) or (B) oleic acid (2 mM), for different times (representative of 2 performed).
HEP_23935_sm_suppFig-S5.tif169KSupplemental Figure 5: The silencing of CD40 inhibits the CD154-induced increased XBP1 mRNA splicing under tunicamycin- and oleic acid-treated HepG2 cells. (A): Fold-induction of CD40 mRNA in HepG2 cell lysates at 48, 72 and 96 hours following transfection with luciferase and CD40 siRNAs (mean ± SD, n=3). (B): Flow cytometry analysis of CD40 expression on HepG2 cells at 72 and 96 hours following transfection with luciferase and CD40 siRNAs (representative of 3 performed with si1CD40, similar results were obtained with si2CD40). (C and D): Fold-induction of the ratio of spliced to unspliced XBP1 mRNAs (XBP1s/u) from HepG2 cell lysates following incubation of HepG2 cells 18 hours with or without TM (C) or 8 hours with or without 2mM OA (D), in the presence or not of rsCD154 and after transfection with luciferase and CD40 siRNAs (mean ± SD, n=3; * p<0.05). Data are normalized to cells grown in respective vehicle medium without rsCD154 and transfected with luciferase siRNA.
HEP_23935_sm_suppFig-S6.tif68KSupplemental Figure 6: CD154 reduces toxicity associated to long term exposure to oleic acid. HepG2 cells were cultured in the presence of 2 mM OA in the presence or not of rsCD154. Cell viability was measured in a sulforhodamin B cytotoxicity assay. The data plotted represent the mean ± SD, n=4, * p<0.05. Hundred per cent viability represents the optical density for untreated cells.
HEP_23935_sm_suppFig-S7.tif73KSupplemental Figure 7: High concentrations of oleic acid inhibit apoB100 secretion in HepG2 cells. ApoB100 concentration in the supernatants of HepG2 cells following 4 and 8 hours incubation with vehicle medium, 1 and 2 mM OA (mean ± SD, n=4, * p<0.05).
HEP_23935_sm_suppFig-S8.tif127KSupplemental Figure 8: Olive oil-rich diet and TM treatment increase the expression of CD154. (A and B): platelet P-selectin expression as measured by flow cytometry in WT and CD154KO mice at time 0 (T0) and following 3 weeks (T=3 weeks) of olive oil-rich diet (A) and in WT and CD154KO mice at time 0 (T0) and 24 hours after TM injection (T=24 h) (B) (mean ± SD, n=5 mice in each group; * p<0.05). (C and D): plasma concentration of soluble CD154 (sCD154) in WT mice at time 0 (T0) and following 3 weeks (T=3 weeks) of olive oil-rich diet (C) and in WT mice at time 0 (T0) and 24 hours after TM injection (T=24 h) (D) (mean ± SD, n=5 mice in each group; * p<0.05). (E and F): CD154 expression on platelets as measured by flow cytometry in WT mice at time 0 (T0) and following 3 weeks (T=3 weeks) of olive oil-rich diet (E) and in WT mice at time 0 (T0) and 24 hours after TM injection (T=24 h) (F) (mean ± SD, n=5 mice in each group; * p<0.05).
HEP_23935_sm_suppFig-S9.tif118KSupplemental Figure 9: (A and B): Impaired IL-6 induction following the olive oil-rich diet in CD154KO mice (A): Mouse plasma concentration of IL-6 in WT and CD154KO mice at time 0 (T0) and following 3 weeks of olive oil-rich diet (T=3 weeks) (mean ± SD, n=5 mice in each group; * p<0.05). (B): Fold-induction of IL-6 mRNA in WT and CD154KO livers from mice fed for 3 weeks on sham diet or olive oil-rich diet (Olive oil diet) (mean ± SD, n=8 mice in each group; * p<0.05). Data are normalized to WT mice in sham diet conditions. (C and D): Fold-induction of IL-6 mRNA in HepG2 pcDNA cell lysates (C), and in HepG2 IRE1 DN cell lysates (D) following incubation with TM or rsCD154 (the data plotted represent the mean ± SD, n=4). Inserts: Fold-induction of IL6 mRNA in HepG2 pcDNA cell lysates (C), and in HepG2 DN IRE1 cell lysates (D) following 12 hours incubation with or without TM and with or without rsCD154 (mean ± SD, n=4; * p<0.05). Data from figures C and D are normalized to cells grown in respective vehicle medium without TM or rsCD154.
HEP_23935_sm_suppTable-S1.doc37KSupporting Table S1
HEP_23935_sm_suppInfo.doc76KSupporting Information

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