Fig 2: 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

Fig 3: PUMA mediates the antitumour effects of gilteritinib in vivo. A, Nude mice were injected s.c. with 5 × 106 WT or PUMA‐KO HCT116 cells. After 1 wk, mice were oral gavaged with 5 mg/kg gilteritinib or the vehicle control for 10 consecutive days. Tumour volume at indicated time‐points after treatment was calculated and plotted with P values, n = 6 in each group. Arrows indicate gilteritinib injection. B, Mice with WT HCT116 xenograft tumours were treated with 5 mg/kg gilteritinib or the vehicle for 5 consecutive days. The levels of indicated proteins in randomly selected tumours were analysed by Western blotting. C, Paraffin‐embedded sections of WT or PUMA‐KO tumour tissues from mice treated as in (B) were analysed by TUNEL staining. D, Paraffin‐embedded sections of WT or PUMA‐KO tumour tissues from mice treated as in (B) were analysed by activated caspase 3 staining. Results in (C) and (D) were expressed as means ± SD of three independent experiments. **P < .01

Fig 4: PUMA is required for the apoptotic and antitumour effects of gilteritinib via mitochondrial pathway. A, PUMA expression was analysed by Western blotting in WT and PUMA‐KO HCT116 cells. B, WT and PUMA‐KO HCT116 cells were treated with 50 nmol/L gilteritinib for 24 h. Apoptosis was analysed by Annexin V/PI staining followed by flow cytometry. C, WT and PUMA‐KO HCT116 cells were treated with 50 nmol/L gilteritinib for 24 h. Cleaved caspase 3 and 9 were analysed by Western blotting. D, Cytosolic fractions isolated from WT and PUMA‐KO HCT116 cells treated with 50 nmol/L gilteritinib for 24 h were probed for cytochrome c by Western blotting. β‐Actin and cytochrome oxidase subunit IV (Cox IV), which are expressed in cytoplasm and mitochondria, respectively, were analysed as the control for loading and fractionation. E, WT and PUMA‐KO HCT116 cells were treated with 50 nmol/L quizartinib or dovitinib for 24 h. Cleaved caspase 3 and 9 were analysed by Western blotting. F, WT and PUMA‐KO HCT116 cells were treated with 50 nmol/L quizartinib or dovitinib for 24 h. Apoptosis was analysed by Annexin V/PI staining followed by flow cytometry. G, Colony formation of WT and PUMA‐KO HCT116 cells treated with 50 nmol/L gilteritinib for 24 h at 10 d following crystal violet staining of attached cells. Upper, representative pictures of colonies; Bottom, quantification of colony numbers. H, SW480 cells transfected with si control or si PUMA were treated with 50 nmol/L gilteritinib for 24 h. Apoptosis was analysed by Annexin V/PI staining followed by flow cytometry. I, SW480 cells transfected with si control or si PUMA were treated with 50 nmol/L gilteritinib for 24 h. Cleaved caspase 3 and 9 were analysed by Western blotting. Results in (B), (F), (G) and (H) were expressed as means ± SD of 3 independent experiments. **P < .01

Fig 5: Runx1 contributes to cardiac hypertrophy by activating p53 signalling pathway. (A) Representative Western blots showing the protein levels of Runx1, p53, Noxa, Bax and Puma in mice heart tissues in the indicated groups, (n = 4 mice per group)