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. 2012 Jul 30;109(33):13325–13330. doi: 10.1073/pnas.1120193109

Fig. 4.

Fig. 4.

ASPP2 inhibits autophagy by binding to ATG5/ATG12, and thus prevents the formation of a complex of ATG5/ATG12/ATG16. (A) Immunofluorescent staining of LC3 (green) in HKe3 ER:HRAS V12 cells with the indicated treatments. TO-PRO-3 (Invitrogen) was used to stain nuclei (blue). Images were taken with a ×63, oil-immersion objective lens. HKe-3 ER:HRAS V12 cells were transfected with control siRNA or siRNA against ASPP1, iASPP (B), or ASPP2 (C) for 3 d, followed by treatment without (control) or with 100 nM 4-OHT for 1 d. (D) Depletion of ASPP1 or ASPP2 promotes amino acid starvation (A.A.S.)-induced autophagy. HCT116 cells were transfected with the indicated siRNA for 4 d, followed by amino acid starvation for 5 h. The graph shows the ratio of LC3II/I. Error bars indicate SD. (E) ASPP2 binds ATG5/ATG12 in HKe3 ER:HRAS V12 cells on RAS activation. Total cell lysates from HKe3 ER:HRAS V12 cells treated without (control) or with 4-OHT (1 d) were immunoprecipitated with an anti-ASPP2 antibody or control IgG. HA, hemagglutinin; IP, immunoprecipitation. (F) Coimmunoprecipitation experiment using in vitro translated ATG5 and V5-tagged ASPP2 fragments. V5 antibody was used to immunoprecipitate ASPP2. ATG5 and ASPP2 levels were then analyzed by SDS/PAGE/immunoblotting using antibodies against ATG5 or V5. Stars indicate the correct size of ASPP2 fragments. The IgG light chain (IgGL) is labeled. (G) ASPP2 depletion enhances the binding between ATG16 and ATG5/ATG12 in HCT116 cells. HCT116 cells were transfected with control siRNA or siRNA against ASPP2 for 4 d. Total cell lysates from HCT116 cells with the indicated treatments were immunoprecipitated with an anti-ATG16 antibody or control IgG. (H) N-terminal ASPP2 competes with ATG16 to bind ATG5 in vitro. In vitro translated ATG16, ATG5, and V5-tagged ASPP2 fragments were mixed. ATG16 antibody was used to immunoprecipitate ATG16. ATG16 and ATG5 levels were then analyzed by SDS/PAGE/immunoblotting using antibodies against ATG16 or ATG5. Stars indicate the correct size of the ASPP2 fragments. (I) Diagram summarizes the role of ASPP2 in regulating autophagic activity, thereby dictating the cellular response to oncogenic RAS (details are provided in Discussion).