Fig. 3. IFN-γ and TNF-α synergise to induce cell death and BCL-G expression in primary human colonic organoids.

a–d Undifferentiated primary human colonic organoids were treated with IFN-γ (10 ng/ml), TNF-α (10 ng/ml) or IFN-γ+TNF-α (10 ng/ml each) for the indicated time points. a Relative organoid viability measured at 24 and 72 h in n = 5 organoid lines. b Representative live images of one of the organoid lines used in panel (a), and treated as indicated. c Relative mRNA expression of BCL-G_S/L measured by RT-qPCR at 4 h after cytokine treatment in n = 4 organoid lines. d Relative mRNA expression of BCL-G_S/L measured by RT-qPCR at 4 and 8 h after combined cytokine treatment in n = 4 organoid lines. e Primary human colonic organoids undifferentiated or differentiated for 3 days were treated with IFN-γ+TNF-α (10 ng/ml each) for 4 h. Heatmap showing ranked relative mRNA expression of differentiation markers (LGR5, MUC2 and VIL1) across n = 3 organoid lines (left). Relative mRNA expression of BCL-G_S/L measured by RT-qPCR in n = 3 organoid lines (right). All organoid lines were generated from colonic biopsies from non-IBD individuals. Data shown are the mean ± S.E.M. ^//p < 0.01 and ^///p < 0.001 (two-way ANOVA followed by Tukey’s multiple comparisons test vs. undifferentiated, non-treated organoids), **p < 0.01 and ***p < 0.001 (two-way ANOVA followed by Tukey’s multiple comparisons test as indicated), ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test as indicated). NT — non-treated, P — patient-derived organoid line.