Figure 5

Downregulation of ASPP2 increases the BECN1 interactome and BECN1-associated PIK3C3 kinase activity in HCC cells. (a) HCC-LM3 and HepG2 infected with LV-shNon or LV-shASPP2, followed by 6 h EBSS treatment, were lysed for immunoprecipitation with BECN1-specific antibody and immunoblotted with antibodies as indicated. (b) HEK293T cells were transfected with Flag-BECN1, HA-UVRAG, HA-ATG14, and ASPP2-V5 or Flag-BECN1, HA-UVRAG, HA-ATG14, and V5-vector for 24 h, and then treated with EBSS for 6 h. Cell lysates were immunoprecipitated with Flag antibody (BECN1) and analyzed by western blotting. (c) HCC-LM3 and Huh7 transfected with pcDNA3.1 or pASPP2, followed by 6 h EBSS treatment, were lysed for immunoprecipitation with BECN1-specific antibody and immunoblotted with antibodies as indicated. (d) HCC-LM3 cells infected with LV-shNon or LV-shASPP2 for 72 h, or transfected with vector or pASPP2 for 48 h, were treated with EBSS for 6 h. Then cell lysates were immunoprecipitated with anti-BECN1 antibody. The PI3-Kinase activity ELISA kit was used to measure the kinase activity of the PIK3C3 protein immunoprecipitated. (e) At 24 h posttransfection with p40(phox)PX-EGFP fusion, HCC-LM3 with LV-shNon or LV-shASPP2 were detected using an inverted fluorescence microscope. Huh7 were detected after co-transfection with p40(phox)PX-EGFP fusion and pASPP2 vectors for 48 h. p40(phox)PX-EGFP-positive vesicles were quantified and analyzed. The scale bars represent 10 μm. Data are shown as the means±S.D. from triplicate experiments. (*P<0.05; **P<0.01)