Extended Data Fig. 2: (related to Fig. 1). VDAC2 deficiency sensitizes tumour cells to IFNγ-induced cell death.
From: VDAC2 loss elicits tumour destruction and inflammation for cancer therapy
a, Control or VDAC2-deficient tumour cell viability after co-culture with sgNTC-, sgIfng-, sgTnf- or sgPrf1-transduced OT-I cells at E:T ratio of 0.5:1 for 24 h (n = 3 per group). b, Control or VDAC2-deficient B16-OVA tumour cell viability after co-culture with OT-I cells (at E:T ratio of 0.5:1) and anti-IFNγ blocking antibody (or isotype; 10 μg ml–1) for 24 h (n = 2 per group). c, Indicated B16-OVA tumour cell viability after co-culture with OT-I cells at E:T ratio of 0.5:1 for 24 h (n = 3 per group). d, Control and VDAC2-deficient B16-OVA tumour cell death after treatment with or without IFNγ (10 ng ml–1) for 24 h (n = 3 per group). e, LDH release from control and VDAC2-deficient B16-OVA tumour cells treated with or without IFNγ (10 ng ml–1) or TNF (10 ng ml–1) for 24 h (n = 3 per group). f, Ametrine+ control or VDAC2-deficient B16-OVA tumour cells were mixed at a 1:1 ratio with mCherry+ spike B16-OVA cells and treated with or without indicated cytokines for 24 h. The relative number of control or VDAC2-deficient cells compared with internal spike cell control is shown (n = 3 per group). g, Immunoblot analysis of pro- (P35) and cleaved (P19 and P17) caspase-3; pro- (P35) and cleaved (P20) caspase-7; or pro- (P53) and activated (P34) GSDME in control or VDAC2-deficient B16-OVA tumour cells treated with IFNγ for indicated timepoints. h, Immunoblot analysis of pro- (P53) and activated (P34) GSDME in indicated B16-OVA tumour cells treated with or without IFNγ for 18 h. i, Expression of caspase-3, caspase-7 and GSDME in indicated B16-OVA tumour cells. j, k, Real-time survival analysis of indicated B16-OVA tumour cells after treatment with 1 ng ml–1 (j) or 10 ng ml–1 (k) of IFNγ (n = 2 per group). l, Control and VDAC2-deficient B16-OVA tumour cell death before or after treatment with IFNγ (10 ng ml–1) plus pan-caspase inhibitor emricasan, ferroptosis inhibitor ferrostatin-1 (Fer-1), necroptosis inhibitor necrostatin-1 (Nec-1) or GSDMD-mediated pyroptosis inhibitor disulfiram for 24 h (n = 3 per group). m, Activated caspase-3+ cells among control (n = 7) and VDAC2-deficient (n = 5) B16-OVA tumours. n, Immunoblot analysis of indicated proteins in control or VDAC2-deficient B16-OVA tumour lysates at day 19 after tumour inoculation. Tumour-bearing mice were treated with isotype IgG or anti-PD-L1 antibody at days 7, 10 and 13 after tumour inoculation (n = 3 per group). Densiometric quantification of cleaved (P19) caspase-3, cleaved (P20) caspase-7 and activated (P34) GSDME is shown. o, IFNγ levels in B16-OVA tumour lysates at day 19 after tumour inoculation. Tumour-bearing mice were treated with isotype IgG or anti-PD-L1 antibody at days 7, 10 and 13 after tumour inoculation (n = 6 per group). p, Relative expression of Vdac1, Vdac2 and Vdac3 in indicated mouse cells from publicly available scRNA-seq dataset (GSE121861), which profiled six syngeneic tumour models including B16-F10 melanoma, EMT6 breast mammary carcinoma, LL2 Lewis lung carcinoma, CT26 colon carcinoma, MC38 colon carcinoma and Sa1N fibrosarcoma. q, Relative expression of VDAC1, VDAC2 and VDAC3 in indicated human cell populations (from melanoma (GSE215121, left) and lung cancer (GSE148071, right) datasets). r, Real-time survival analysis of indicated B16-OVA tumour cells after treatment with 1 ng ml–1 (left; n = 2 per group) or 10 ng ml–1 (right; n = 2 per group) of IFNγ. The same control and VDAC2-deficient B16-OVA tumour cells are presented in Fig. 1l. Data are representative of three (d, m), two (a, c, f–l, n) or one (b, e, o, r) independent experiments and are mean ± s.e.m. Two-tailed unpaired Student’s t-test (m, o). Two-way ANOVA (a, c–f, l).
