Figure 5.

Sunitinib induces TP53 autophagic degradation in an SQSTM1-dependent manner. (a) Representative western blots show the effect of the knockdown of SQSTM1 on the sunitinib-induced degradation of TP53. Cells were treated with 7.5 µM sunitinib for 24 h in the absence or presence of 10 µM CQ for 2 h before the addition of sunitinib. The TP53 or SQSTM1 expression levels from biological triplicates were normalized with GAPDH and quantified with Quantity One. The data represent the average of 3 independent experiments. Each value represents a ratio of the maximal level of TP53 or SQSTM1. (b) The colocalization of TP53 with SQSTM1 in untreated or sunitinib-treated HCT116 cells. The cells were stained for TP53 (green), SQSTM1 (red) and nuclei (blue). Scale bar: 20 µm. (c) HCT116 cells were treated with sunitinib for 24 h in the absence or presence of CQ for 2 h prior to the addition of sunitinib. The cell lysates were subjected to immunoprecipitation using an antibody against TP53, followed by western blots to detect TP53 and SQSTM1 expression. (d) 293FT cells were transfected with GFP-HMGB1, FLAG-SQSTM1 and GFP-TP53 and were then treated with sunitinib and CQ for 24 h. The cell lysates were subjected to immunoprecipitation using an antibody against FLAG, followed by western blots to detect TP53, FLAG and HMGB1 expression. (e) HCT116 cells and SQSTM1 knockdown HCT116 cells were treated with sunitinib plus CQ. The cell lysates were subjected to immunoprecipitation using an antibody against TP53, followed by western blots to detect HMGB1, TP53 and SQSTM1 expression. SUNI, sunitinib; CQ, chloroquine; WCL, whole cell lysates. All experiments were performed in triplicate. For western blots, data from 1 of 3 experiments are shown.