Supplementary Figure legends
Supplementary Figure S1: Characterization of the SW480 Fas WT , SW480 Fas C199V and SW480 lacZ cells. (a) Fas cell surface expression was assessed by FACS analysis on the 3 stable lines, Fas expression on SW480 lacZ cells representing endogenous Fas level. (b) Fas total expression was investigated by western blotting using several antibodies directed against Fas recognizing both endogenous and transfected V5-tagged Fas. V5 antibody was used to detect exclusively the transfected V5-tagged Fas.
Supplementary Figure S2: (a) DHHC7 localizes in the Golgi apparatus. SW480 cells transfected with vectors coding for DHHC7-HA or the fusion protein Golgi-EYFP were immunostained with DHHC7 antibody and phalloidin coupled to AlexaFluor 647 before confocal analysis. Images are representative of cells typically observed in the indicated conditions. (b) Co-immunoprecipitation of overexpressed Fas and DHHC7. SW480 Fas WT and SW480 Fas C199V cells were transiently transfected with vectors coding for AcGFP alone (EV), or the fusion proteins DHHC7-AcGFP and DHHC7 C160S-AcGFP and submitted to Fas immunoprecipitation. After SDS-PAGE, the co-immunoprecipitation of DHHC7-AcGFP and DHHC7 C160S-AcGFP was detected by immunoblotting with anti DHHC7 antibody.
Supplementary Figure S3: Palmitoylation regulated Fas surface expression. (a) SW480 cells transfected with siRNA control or targeting DHHC7 were analysed by flow cytometry for TNFR1 surface expression. (b) The CRC cells SW48, HCT15 and DLD1 transfected with siRNA control or targeting DHHC7 were analysed by flow cytometry for Fas surface expression. (c-d) SW480 transfected with siRNA targeting DHHC7 (c) or treated for 16 hours with bromopalmitate were submitted to protein biotinylation protocol to detect Fas expressed at cell surface. (e) SW480 Fas WT and SW480 Fas C199V treated for 16 hours with bromopalmitate were analysed by flow cytometry for Fas surface expression. Quantification of Fas surface expression by flow cytometry represents the median of DX2-PE fluorescence (mean of 4 experiments +/- SD).
Supplementary figure S4: DHHC7 expression increases Fas surface expression in SW480 Fas WT cells. Protein surface biotinylation assay was performed on SW480 Fas WT cells non transfected or transfected for 48 hours with 1 or 2 µg of plasmid coding for DHHC7-HA and analysed by immunoblotting with indicated antibodies.
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