Figure 4.

Activation of p65 mediates PUMA induction in response to gilteritinib treatment. A, HCT116 cells were treated with gilteritinib for 24 h at indicated concentration. Indicated proteins were analysed by Western blotting. B, HCT116 cells were treated with 50 nmol/L gilteritinib at indicated time‐points. Expression of p‐p65 (S536) and p65 was analysed by Western blotting. C, HCT116 cells were transfected with either a control scrambled siRNA or a p65 siRNA for 24 h and then treated with 50 nmol/L gilteritinib for 24 h. p65 and PUMA expressions were analysed by Western blotting. D, HCT116 cells were treated with 10 μmol/L BAY11‐7082 for 1 h, and then with 50 nmol/L gilteritinib for 24 h. Nuclear fractions were isolated from cells and analysed for p65 expression by Western blotting. Lamin A/C and β‐actin, which are expressed in nucleus and cytoplasm, respectively, were used as controls for loading and fractionation. E, HCT116 cells were treated with 10 μmol/L BAY11‐7082 for 1 h, and then with 50 nmol/L gilteritinib for 24 h. The level of p‐p65 (S536) and PUMA was analysed by Western blotting. F, Chromatin immunoprecipitation (ChIP) was performed using anti‐p65 antibody on HCT116 cells following gilteritinib (50 nmol/L) treatment for 12 h. The IgG was used to control for antibody specificity. PCR was carried out using primers surrounding the p65 binding sites in the PUMA promoter