Fig 2: TCF4 inhibition sensitizes GC cells to cisplatin-induced ferroptosis in vitro.a Cell viability of indicated GC cells following treatment with cisplatin (AGS and MKN-45 for 100 µM, HGC-27 for 10 µM) and in the absence or presence of ferrostatin-1 (2 µM) or liproxstatin-1 (1 µM) for 24 h. b MDA production in GC cells followed by treatment with cisplatin (AGS and MKN-45 for 10 µM, HGC-27 for 1 µM). c Cell viability of GC cells expressing sgNC or sg-TCF4 and treated with different concentrations of cisplatin for 24 h. d Cell viability of GC cells transfected with control or TCF4-coding plasmid and treated with different concentrations of cisplatin for 24 h. e, f 4-HNE production in TCF4-KO (e) or overexpressing (f) GC cells treated with cisplatin (AGS and MKN-45 for 10 µM, HGC-27 for 1 µM). Data are presented as the mean ± SD of three independent experiments. The p-values in panels a, e, f, were calculated by one-way ANOVA. The p-values in panels c, d, were calculated by two-way ANOVA. The p-values in panel b were calculated by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: TCF4 negatively modulates ferroptosis through GPX4 targeting.a Venn diagram showing genes at the intersection of TCF4 bound promoters that were identified by ChIP-seq and previously reported ferroptosis-related genes. b Bar chart showing log2FC of the expression of five genes in TCF4 siRNA transfected HGC-27 cell. c Relative gene expression levels of GPX4, GCLM, CRYAB, LPCAT3, and FDFT1. d Q-PCR analysis of GPX4 mRNA expression in paired GC tissues and adjacent normal tissues (n = 34). e Protein expression of GPX4 in GC tissues (T) and adjacent normal tissues (N). f, g IHC staining (f) and H-score (g) for GPX4 in adjacent normal tissues (normal, n = 9) and GC tissues (tumor, n = 9). Scale bars: 200 µm (insets 50 µm). h, i IHC staining (h) and H-score (i) for GPX4 in N (normal tissues, n = 21), SG (superficial gastritis, n = 21), AG with IM (atrophic gastritis with intestinal metaplasia, n = 21), DYS (dysplasia, n = 25), and GC (n = 21) samples. Scale bars: 200 µm (insets 50 µm). j The overall survival for GC patients was analyzed using Kaplan–Meier curves (log-rank test; n = 384). k GPX4 protein expression in sgNC or sg-GPX4 expressing GC cells. l MDA production in GC cells expressing sgNC or sg-GPX4 and transfected with the TCF4-coding plasmid. m, n Colony formation of GC cells expressing sgNC or sg-GPX4 and transfected with the TCF4-coding plasmid. Data are presented as the mean ± SD of three independent experiments. The p-values in panels c, i, l, n were calculated by one-way ANOVA. The p-values in panels d, g were calculated by Student’s t-test. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: TCF4 deficiency or Wnt signaling inhibition promote cisplatin sensitivity through ferroptosis in vivo.a Schematic description of the experimental design used to establish the animal model. b, c Tumor weight (b) and tumor growth curves (c) in the nude mouse xenograft model. d, e IHC staining (d) and H-score (e) for 4-HNE in xenografts. Scale bars: 200 µm (insets 50 µm). f MDA production in tumor tissues. g Schematic description of the experimental design used to establish the animal model. h, i. Tumor weight (h) and tumor growth curves (i) in the nude mouse xenograft model. j, k IHC staining (j) and H-score (k) for 4-HNE in xenografts. Scale bars: 200 µm (insets 50 µm). l MDA production in tumor tissues. Data are presented as the mean ± SD of 6 biologically independent animals. All p-values were calculated using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: TCF4 acts as a repressor of ferroptosis in GC cells.a Cell viability of GC cells expressing sgNC or sg-TCF4 treated with different concentrations of erastin for 24 h. b Cell viability of GC cells transfected with control or TCF4-coding plasmid and treated with erastin for 24 h. c Cell death measurement of AGS expressing sgNC or sg-TCF4 treated with erastin (20 µM) in the absence or presence of ferrostatin-1 (2 µM), liproxstatin-1 (1 µM), Z-VAD-FMK (10 µM), necrosulfonamide (0.5 µM), or 3-methyladenine (250 µM) for 24 h. d Cell viability of AGS expressing sgNC or sg-TCF4 treated with erastin (50 µM) in the absence or presence of ferrostatin-1 (2 µM), liproxstatin-1 (1 µM), Z-VAD-FMK (10 µM), necrosulfonamide (0.5 µM), or 3-methyladenine (250 µM) for 24 h. e MDA production in GC cells expressing sgNC or sg-TCF4. f MDA production in GC cells transfected with control or TCF4-coding plasmid. Data are presented as the mean ± SD of three independent experiments. The p-values in panels a–d were calculated by two-way ANOVA. The p-values in panel e were calculated by one-way ANOVA. The p-values in panle f were calculated by Student’s t-test and one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.