Fig. 1.
A Representative dot-plots showing the percentages of AV- and/or PI-positive HCT116 cells either left untreated or transfected with a control or hepcidin siRNA for 48 h in the presence or absence of exogenous hepcidin (HAMP) (1000 ng/mL). One of 5 separate experiments in which similar results were obtained is shown. B, C SYTOX green uptake and levels of HMGB1 in the culture supernatants of HCT116 cells either left untreated or transfected with a control or hepcidin siRNA (25 nmol/L) for 48 h. Data are expressed as mean ± SD of three experiments. ****p < 0,0001. D Representative Western blots showing the full length (F-L) and cleaved (N-T) GSDM E and β-actin in HCT116 cells either left untreated or transfected with a control or hepcidin siRNA (25 nmol/L) for 48 h. E Histograms showing the percentages of AV- and/or PI-positive HCT116 cells transfected with either a hepcidin siRNA (25 nmol/L) or co-transfected with control siRNA/hepcidin siRNA (25 nmol/L) plus GSDM-E siRNA (5 nmol/L) for 48 h. Data are expressed as mean ± SD of all experiments. Hepcidin siRNA vs hepcidin siRNA plus GASDM-E siRNA, **p < 0.01; ****p < 0,0001. F Heat map showing microarray-based differential expression, log2 (fold change) of TNF-related genes in HCT116 transfected with a control or hepcidin siRNA (25 nmol/L) for 48 h. G Histograms showing the levels of TNF protein in HCT116 either left untreated or transfected with a control or hepcidin siRNA (25 nmol/L) for 48 h; ****p < 0,0001. H Representative Western blots showing F-L GSDM E, N-T GSDM E, and β-actin. One of 4 separate experiments in which similar results were obtained is shown. I Histograms showing the percentages of AV- and/or PI- positive HCT116; data are expressed as mean ± SD of all experiments. *p < 0,05. J Representative images and relative graphs showing the volume of CT26-derived tumors in BALB/c mice. CT26 cells were transfected with either a control siRNA or hepcidin siRNA (25 nmol/L) for 36 h and subcutaneously injected into the left flank of mice (1 × 106 per mouse) (day 0). Tumor growth was monitored until sacrifice (day 13). Each point in the graph represents the value of the tumor volume in each mouse. **p < 0,01. K Representative dot-plots showing the percentages of CD3+ CD8+ and CD3+ CD8− cells from CT26-derived tumors. One of 2 experiments in which 8 mice per group were analyzed is shown. L Representative images and the relative graph showing the volume of CT26-derived tumors. CT26 were transfected with either a control siRNA or hepcidin siRNA (25 nmol/L) for 36 h and subcutaneously injected into the left flank of BALB/c mice (1 × 106 per mouse) (day 0). CD8+ cell depletion was made with intraperitoneal injection of α-CD8 (100 µg per mouse). Tumor growth was monitored until sacrifice (day 13). Each point in the graph represents the value of tumor volume in each mouse. **p < 0,01. M Tumor incidence following injection of CT26 or TS/A cells in mice that were previously vaccinated with hepcidin siRNA-transfected CT26 cells. Mitomycin-treated cells were injured at the same of vaccination as a positive control. N The percentage of mice free of tumors at day 26 after the vaccination protocol as described in L and injected again into the right flank with CT26. O Representative images and the relative graph showing the volume of CT26-derived tumors in BALB/c mice injected with CT26 cells transfected with either a control or hepcidin siRNA (25 nmol/L) for 36 h and then subcutaneously injected into the left flank. PD-1 blockade was made with intraperitoneal injection of α-PD-1 (100 µg per mouse). Each point in the graph represents the value of tumor volume in each mouse; *p < 0,05; ***p < 0.001