Supplementary Figure 1. ASPP2 overexpression-inducedCHOPmediates apoptotic cell death in hepatoma cell lines.Hep1-6, HepG2, Hep3B and Huh7 cells were transfected with the ASPP2 plasmid (ASPP2 Pl) for 24 hours.(a) An immunofluorescence assay showed that ASPP2 overexpression induced CHOP expression in HepG2, Hep3B and Huh7 cells. (b) Annexin V/PI was used to detect apoptosis in Hep1-6, HepG2, Hep3B and Huh7 cells after transfection with the ASPP2 Pl for 12 and 24 hours. The Annexin V+/PI- cells are apoptotic cells and representative dots plots are shown in left panel; right panel is the mean values of percentages of apoptotic cells.(c) MTT assay was used to detect cell viability in Hep1-6, HepG2, Hep3B and Huh7 cells after transfection with the ASPP2 Pl for 12 and 24 hours. (d) HepG2 cells were transfected with the ASPP2 Pl with or without co-transfection with CHOP siRNA (CHOP si). Immunoblot assay was used to detect the level of PARP (left panel). The ratio of p85 to β-actin was calculated from densitometry scanningdata (right panel).All data are shown as the mean ± SEM of three independent experiments in (a), (b), (c) and (d). (e) HepG2 cells were transfected with the ASPP2 Plwith or without co-transfection with p53 siRNA (p53 si). Immunoblot assay was used to detect the levels of LC3 I/II, p62, PARP, DRAM and CHOP. (f) Hep3B cells were transfected with the ASPP2 Plwith or without co-transfection with CHOP/p73/DRAM siRNA (CHOP si/p73 si/DRAM si). Immunoblot assay was used to detect the levels of proteins with indicated antibodies. (g) Hep1-6 cells were transfected with the ASPP2 Plwith or without co-transfection with p53 si. Immunoblot assay was used to detect the levels of proteins with indicated antibodies.
Supplementary Figure 2. CHOP mediates ASPP2 overexpression-induced autophagy. (a) Hep1-6 cells were transfected with the ASPP2 plasmid (ASPP2 Pl) with or without pre-treatment with Bafilomycin A1 (BafA 1). Immunoblot assay was used to detect the effect of BafA1 treatment on inhibiting ASPP2-induced autophagy. (b) Hep1-6 cells were transfected with the ASPP2 Pl with or without co-transfection with CHOP siRNA (CHOP si). Immunoblot assay was used to detect the effect of CHOP knockdown on inhibiting ASPP2-induced autophagy. (c and d) Immunoblot assay was used to detect the effect of BafA 1 pre-treatment (c) or CHOP knockdown via CHOP si (d) on the expression of indicated proteins. (e and f) Huh7 cells were transfected with the GFP-LC3 plasmid and ASPP2 Pl, andtargeted siRNA was used to inhibit CHOP expression. Representative images of GFP-LC3 II puncta are shown. Magnification, 1000×. (e). (f) Quantification of autophagosome-developed cells in which more than ten GFP-LC3 II puncta could be observed. The data are shown as the mean ± SEM of three independent experiments. (g) Hep1-6 cells were transfected with the ASPP2 Pl with or without pre-treatment with BafA 1. Immunoblot assay was used to detect the level of apoptosis with indicated antibodies.
Supplementary Figure 3. Fluorescence microscopyassay. (a) Detection of the co-location of Bcl-2 (green) and Beclin-1 (red) in Hep1-6 cells with or without transfection of the ASPP2 plasmid. DAPI was used to stain the nucleus. Magnification, 1000×.(b) Hep1-6 cells were transfected with the GFP-LC3 plasmid or co-transfected with the indicated plasmids (including the ASPP2 plasmid and the Bcl-2 plasmid) and Beclin-1 siRNA. Fluorescence microscopy was then used to detect the co-localization of ASPP2 (blue), GFP-LC3 puncta (green) and Beclin-1(red). Magnification, 1000×.(c) Hep1-6 cells were transfected with the ASPP2 plasmid with or without co-transfection with Bcl-2 plasmid. Fluorescence microscopy was then used to detect the co-localization of ASPP2 (blue), Bcl-2 (green) and Beclin-1(red). Magnification, 1000×.
Supplementary Figure 4.ASPP2-induced CHOP does not affect AKT/mTOR axis or induce TRIB3 expression and TRIB3 does not bind to AKT.(a) Hep1-6 cells were transfected with the ASPP2 plasmid (ASPP2 Pl) with or without co-transfection with CHOP siRNA (CHOP si). Immunoblot assay was used to detect AKT/p-AKT, mTOR/p-mTOR, TRIB3 expression. (b) Hep1-6 cells were transfected with theASPP2 Plfor 24 hours. Anti-TRIB3 antibody was used to immunoprecipitate TRIB3. TRIB3 and AKT were then analyzed by immunoblot assay.
Supplementary Figure 5. CHOP does not directly bind to ASPP2, Bcl-2 and Beclin-1. Hep1-6 cells were transfected with the ASPP2 plasmid (ASPP2 Pl) for 24 hours and then nuclei were extracted. Anti-CHOP antibody was used to immunoprecipitate CHOP in the extracted nuclei. Total cell lysates were used as input or were immunoprecipitated with control IgG as indicated. ASPP2, Bcl-2 and Beclin-1were then analyzed by immunoblot assay.
Supplementary Figure 6.DRAM does not affect the interaction of Bcl-2 with Beclin-1 or the interaction of ASPP2 with Bcl-2. Hep1-6 cells were transfected with the ASPP2 plasmid (ASPP2 Pl) for 24 hours with or without co-transfection with DRAM siRNA (DRAM si). Nucleus (a) and nucleus-free cytoplasm (b) were extracted.Anti-ASPP2 antibody was used to immunoprecipitate ASPP2in extracted nuclei (a) and anti-Beclin-1 antibody was used to immunoprecipitate Beclin-1 in nucleus-free cytoplasm (b). Then Bcl-2 was analyzed by immunoblot assay.
Supplementary Figure 7.SimultaneousKnockdown of p53/p73 and CHOP produces a stronger inhibition of autophagy and apoptosis. Hep1-6 and Hep3B cells were transfected with the ASPP2 plasmid (ASPP2 Pl)with or without co-transfection with CHOP/p53/p73 siRNAs (CHOP si/p53 si/p73 si). Immunoblot assay was used to detect the levels of proteins with indicated antibodies.