Figure 4.

Sunitinib promotes the interaction of TP53 and HMGB1 and the nucleus-to-cytoplasm transport of HMGB1. (a) 293FT cells were transfected with plasmids encoding FLAG-HMGB1 and GFP-TP53 and were then treated with 7.5 µM sunitinib for 24 h in the absence or presence of 10 µM CQ for 2 h prior to the addition of sunitinib. The cell lysates were subjected to immunoprecipitation using an antibody against FLAG, and the resulting immunoprecipitate was analyzed by TP53 and FLAG immunoblots. (b) The cell lysates from the treated HCT116 cells were subjected to an immunoprecipitation using an antibody against TP53, followed by TP53 and HMGB1 western blots. Cells were treated with 10 µM CQ for 2 h before the addition of 7.5 µM sunitinib for 24 h. (c) Representative western blots show the cytoplasmic (Cyto) and nuclear (Nuc) distribution of HMGB1 in untreated or sunitinib-treated HCT116 cells for 24 h. (d) Immunocytochemistry for HMGB1 and nuclear staining in untreated or sunitinib-treated HCT116 cells. Cells were treated with 7.5 µM sunitinib for 24 h in the absence or presence of CQ for 2 h prior to the addition of sunitinib. Scale bar: 20 μm. (e) Immunocytochemistry for HMGB1 and nuclear staining in untreated or sunitinib-treated TP53^−/− HCT116 cells. Cells were treated with 7.5 µM sunitinib for 24 h. Scale bar: 20 µm. (f) Representative western blots show the effect of HMGB1 cytoplasmic transport following LPS treatment on the degradation of TP53 in HCT116 cells. Cells were treated with LPS for 24 h in the absence or presence of 10 µM CQ for 2 h before the addition of LPS or in the absence or presence of 10 µM MG132 for 8 h before the end of the experiments. SUNI, sunitinib; CQ, chloroquine. All experiments were performed in triplicate. For western blots, 1 of 3 similar experiments is shown.