Fig 2: IFN-? and TNF-a kill IECs independently of mitochondrial apoptosis regulators.A HT-29 cells were pretreated for 1 h with DMSO, caspase-9 inhibitor (Z-LEHD, 10 µM) or zVAD (20 µM), followed by IFN-?/TNF-a (10 ng/ml each). SYTOX Green/Annexin V signals and caspase-3 activity (heatmap) were recorded at indicated times, and relative viability was measured at 72 h (inner graph). B Relative viability of HT-29 cells transfected with siCtrl, siBAK1 or siBAX, followed by IFN-?/TNF-a (10 ng/ml each) for 72 h (left). Western blot validation of BAK1/BAX knockdown (right). C Relative viability of HT-29 cells transfected with siCtrl or siBAK1/BAX, subsequently pretreated for 1 h with DMSO, JAK1-selective compound (upadacitinib), JAK2-selective compound (BMS-911543) or dual JAK1/2 inhibitors (ruxolitinib or baricitinib) at 1 µM, followed by IFN-?/TNF-a (10 ng/ml each) for 72 h. D Relative viability of HT-29 cells pretreated for 1 h with DMSO, baricitinib (1 µM) or a cocktail of apoptosis/necroptosis inhibitors (necrostatin-1s (10 µM), GSK'872 (10 µM), NSA (1 µM) and zVAD (20 µM)) and increasing concentrations of N-acetylcysteine (NAC, 0.1, 1 and 10 mM), followed by (left) IFN-?/TNF-a (10 ng/ml each) or (right) H2O2 (1 mM) for 72 h. Data were mean ± SEM of n = 3 independent experiments. *p < 0.05 and ***p < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). NT non-treated, nec-1s necrostatin-1s, G'872 GSK'872, CASP9-i caspase-9 inhibitor, UPA upadacitinib, BARI baricitinib, RUXO ruxolitinib, BMS BMS-911543, NAC N-acetylcysteine.

Fig 3: The effects of genistein on EGFR expression and the reactivity of EsC cells to EGF. (A–C) EGFR expression in Eca-109 cells treated with different concentrations of genistein for 72 h was detected through qPCR and western blotting. EGFR expression in xenograft mice treated with (D–F) 5 mg/kg or (G–I) 10 mg/kg genistein for 42 d was significantly lower than that in an un-treated group. (J–L) EGFR expression in Eca-109 cells treated with 8 μM of genistein for 9 d. (M) The effect of recombined EGF (20 ng/mL) treatment alone on the proliferation of Eca-109 cells was detected through a CCK-8 assay. (N) The cell viability of Eca-109 cells incubated with genistein (8 μM) for 9 d following treatment with recombined EGF (20 ng/mL) for 24 h, 48 h or 72 h. (O) The reactivity of Eca-109 cells treated with genistein (8 μM) to recombined EGF. (P) A schematic working model to illustrate that genistein inhibits EGFR and its downstream pathways, the PI3K-Akt and JAK1/2-STAT3 signaling pathways, leading to apoptosis and cell cycle arrest in EsC cells. All in vitro experiments were independently repeated three times. The difference between two groups was tested using the Student’s t-test, and comparisons among multiple groups were performed using one-way ANOVA with Dunnett’s test. Data are presented as the mean ± SD. *P<0.05; **P<0.01; Gen, genistein.

Fig 4: Intracellular molecular mechanisms for the serotonin actions on metabolism of MCF-7 cells. a Pyruvate kinase and b succinate dehydrogenase activities in the presence of 5-HT and several inhibitors and antagonists. Plotted values represent the means ± S.E.M. of six independent experiments (n = 6). *P < 0.05 as compared with control. #P < 0.05 as compared with 5-HT. One-way ANOVA followed by Tukey’s post test. Quantification (c) and representative western blot (d) of Jak1 phosphorylation. Quantification (e) and representative western blot (f) of STAT3 phosphorylation. g LDHB mRNA expression. h MCT1 mRNA expression. i Quantification of CREB phosphorylation. j Representative western blot for CREB phosphorylation. k Quantification of PGC1a expression. l Representative western blot for PGC1a expression. For c–l, plotted values represent means ± S.E.M. of seven independent experiments (n = 7). * and #P < 0.05 as compared with control or to 10 µM 5-HT, respectively (Student’s t test for panel E and one-way ANOVA followed by Tukey’s post test for panels c, g, h, i, k). m Glucose consumption. n Lactate production. o Lactate production/glucose consumption ratio. p Mitochondrial activity. For m–p, plotted values represent means ± S.E.M. of five independent experiments (n = 5; * P < 0.05 as compared with control; #P < 0.05 as compared with 5-HT; one-way ANOVA followed by Tukey’s post test)

Fig 5: Targeted in vitro CRISPR screen identifies ANKRD52 as a direct modulator of cancer sensitivity to T cell cytotoxicity.a Schematic overview of CRISPR screen using co-culture of OT-I T cell with MC38-OVA cells. b Cell viability of MC38-OVA cells co-cultured with OT-I T cells during 2.5 days (n = 3 per group per timepoint). Data are represented as mean ± s.d., ****P < 0.0001, significance is determined using multiple two-tailed Student’s t-test. c–e Distribution histograms of log2FC values for all ten sgRNAs targeting Jak1 (c), B2m (d), or Ankrd52 (e) (Cut-off: FC > 1.4, P < 0.05 for enrichment, analyzed by edgeR). f Killing of MC38-OVA cells with indicated sgRNAs by OT-I T cells at indicated ratio in co-culture. Data are representative of three independent experiments and represented as mean ± s.e.m., **P < 0.01, ***P < 0.001, significance was determined using multiple two-tailed Student’s t-test (sgNT #1 vs sgAnkrd52 #1, P = 3.72×10-3 for 1:3, P = 1.74×10-3 for 1:5; sgNT #2 vs sgAnkrd52 #1, P = 3.64×10-3 for 1:3, P = 1.67×10-3 for 1:5; sgNT #1 vs sgAnkrd52 #2, P = 2.28 × 10-4 for 1:3, P = 4.43 × 10-3 for 1:5; sgNT #2 vs sgAnkrd52 #2, P = 2.7 × 10-4 for 1:3, P = 4.57 × 10-3 for 1:5). See also Supplementary Figs. 6 and 7. Source data are provided as a source data file.