Figure 4. PTP4A3 promotes SQSTM1 degradation and serves as a substrate in autophagy. (A) A2780 cells were infected with stable shRNA knockdown constructs against nonspecific targets (shScr) or PTP4A3 (shPTP4A3). Exponentially growing A2780-shScr and A2780-shPTP4A3 cells were lysed and analyzed for stable-state SQSTM1 and PTP4A3 expression levels by western blotting. (B) A2780-shScr and A2780-shPTP4A3 cells were treated with CQ for indicated times before lysis and western blotting analysis of SQSTM1 expression levels. The band intensity ratio of SQSTM1/GAPDH was quantified as described in Materials and Methods, and presented as a histogram in the right panel (mean ± S.D.). (C) CHO-Con, CHO-PTP4A1, and CHO-PTP4A3 stable cells were analyzed for stable-state SQSTM1 expression levels by western blotting. (D) CHO-Con, CHO-PTP4A1, and CHO-PTP4A3 cells were cultured in full medium in the absence (control) or presence (CQ) of CQ for 24 h before lysis and western blot analysis of the indicated proteins. The ratio of SQSTM1/GAPDH was quantified as described in Materials and Methods, and presented as a histogram in the right panel (mean ± S.D.). (E) CHO-Con, CHO-PTP4A1, and CHO-PTP4A3 cells were cultured in full media (control) or in HBSS for 24 h prior to lysis and western blotting analysis of the indicated proteins. (F) CHO-PTP4A3, A2780-PTP4A3, and HCT116 cell lines were cultured in full media in the absence (control) or presence (CQ) of CQ for 24 h prior to lysis and western blotting analysis of the indicated proteins. (G) ATG5 was knocked down using shRNA in A2780-PTP4A3 and HCT116 cells. Exponentially growing cells (in full media) were lysed for western blotting analysis with the indicated antibodies. GAPDH was used as loading control.