Fig 2: Effects of DCM fraction treatment on the expression of apoptosis-related proteins in HT-29 cells. HT-29 cells were incubated with various concentrations of the DCM fraction for 20 h. The expression levels apoptosis-related proteins Fas, pro-caspase-8, Bcl-2, Bcl-xL, Bad, Bax, pro-caspase-9, pro-caspase-7, pro-caspase-3, XIAP, cIAP-1 and cIAP-2 were then determined by western blotting. ß-actin was used as the loading control. The bands were semi-quantitatively analyzed by ImageJ software and the relative protein expression levels are presented. All results were normalized to the untreated control (0 µg/ml). Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. 0 µg/ml DCM. DCM, dichloromethane; xL, extra-large; XIAP, X-linked inhibitor of apoptosis protein; cIAP-1, cellular inhibitor of apoptosis protein-1; cIAP-2, cellular inhibitor of apoptosis protein-2.

Fig 3: (A) Proliferation arrest and clonogenic survival was measured upon exposure up to 100 µM TMZ by colony formation assay in U87MG and A172 cells. Cell death (B) and cell cycle distribution (C) were measured 144 h upon exposure to 50 µM TMZ by flow cytometry using PI staining in A172 cells. (D) Senescence was measured by flow cytometry using SA-ß-Gal staining 144 h after exposure to TMZ in A172 cells. (E) U87MG and A172 cells were treated with 50 µM TMZ. Then, 120 and 144 h later, expression of c-IAP2 and Bcl-2 protein was measured by immunodetection. (A–D) Experiments were performed in triplicate, and differences between treatment and control were statistically analyzed using Student’s t-test (not labeled = not significant, * p < 0.1, ** p < 0.01, *** p < 0.001).

Fig 4: cIAP2 is responsible for H. pylori infection-mediated apoptosis resistance.a AGS cells were infected with H. pylori G27 for 1 h, and the expression of indicated genes was analyzed by qRT-PCR. b, c AGS cells were infected with H. pylori G27 for the indicated time points. mRNA levels of BIRC3 were analyzed by qRT-PCR (b) and cell lysates were analyzed by immunoblotting for indicated proteins (c). b AGS cells pre-treated with BV6 (5 µM) for 1 h were infected with H. pylori G27 for another 1 h, followed by treatment with Raptinal (10 µM) for 2 h. Levels of indicated proteins were analyzed by immunoblotting, n = 3. e Percentage of apoptotic cells from (c) was analyzed by flow cytometry with Annexin V-FITC/PI double staining. f AGS cells transfected with two different siRNAs against BIRC3 or control siRNA were infected with H. pylori G27 for 1 h, followed by treatment with Raptinal (10 µM) for 2 h. Levels of indicated proteins were analyzed by immunoblotting. All data are shown as the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001, n.s.: not significant.

Fig 5: HPV suppresses the expression of RIPK3. (A–C) Two independent uninfected KC cultures (HVK#1, HVK#2) and two HPV16 + KC cultures (HVK16, HPV16) were stimulated with 50 IU/ml IFN? and 30 ng/ml TNFa for the indicated times. The protein expression levels of (A) TRAF2, cIAP1, cIAP2, XIAP, RIP1; (B) FLIP, CYLD, caspase-8 (57 kD), cleaved caspase-8 (41 kD, 43 kD), FADD; and (C) RIPK3, MLKL and MLKL-phospho S358 as detected by Western blotting (WB) in whole cell extracts. ß-actin served as loading control. (D) Two independent uninfected KC cultures (HVK#1, HVK#2) and two HPV16 + KC cultures (HVK16, HPV16) were stimulated with 50 IU/ml IFN? and 30 ng/ml TNFa for 24 h after which the expression levels of cIAP2, FADD, RIPK3, CFLAR, MLKL, and TRAF2 were determined by RT-qPCR. Gene expression was normalized against GAPDH mRNA levels and standardized against the non-stimulated HVK#1. Similar results were observed in two independent experiments. (E) HPV16 E2 expression in HPV + KCs transfected with control siRNA (siControl) or siRNA targeting HPV16 E2 (siE2) stimulated with or without 50 IU/ml IFN? and 30 ng/ml TNFa for 24 h. E2 expression was analyzed by RT-qPCR. Gene expression was normalized against GAPDH mRNA levels and standardized against siControl. Similar results were observed in three independent experiments. ***P < 0.001 and ****P < 0.0001. (F) Expression of cIAP2, FADD, and RIPK3 in hrHPV + KCs transfected with control siRNA (siControl) or siRNA targeting HPV16 E2 (siE2) stimulated with or without 50 IU/ml IFN? and 30 ng/ml TNFa for 24 h. Gene expression was normalized against GAPDH mRNA levels and standardized against siControl. Similar results were observed in two independent experiments. *P < 0.05, ****P < 0.0001.