Extended Data Fig. 9: (related to Fig. 4). Co-targeting APAF-1 or caspase-9 further enhanced STING activation in IFNγ-stimulated VDAC2-deficient tumour cells.
From: VDAC2 loss elicits tumour destruction and inflammation for cancer therapy
a, Real-time survival analysis of control, VDAC2-deficient, APAF-1-deficient, caspase-9-deficient, VDAC2 and APAF-1 co-deficient or VDAC2 and caspase-9 co-deficient 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 Extended Data Fig. 7b. b, c, Cell death (b) or LDH release (c) of control, VDAC2-deficient, BAK-deficient, APAF-1-deficient, caspase-9-deficient, VDAC2 and BAK co-deficient, VDAC2 and APAF-1 co-deficient or VDAC2 and caspase-9 co-deficient B16-OVA tumour cells treated with IFNγ (10 ng ml–1) for 24 h (n = 3 per group). d, e, Cell death of control and VDAC2-deficient B16-OVA tumour cells treated with IFNγ (10 ng ml–1) plus indicated pan-caspase inhibitors (or vehicle) for 24 h (d; n = 3 per group) or 72 h (e; n = 3 per group). f, g, Cell death (f) or LDH release (g) of indicated B16-OVA cells treated with IFNγ (10 ng ml–1) for 72 h (n = 3 per group). h, Immunoblot analysis of indicated proteins in indicated B16-OVA tumour cells treated with or without IFNγ for 24 h. Densiometric quantification of p-TBK1 is shown. i, Relative Ifnb1 levels (versus non-treated control cells) in indicated B16-OVA tumour cells treated with IFNγ for 24 h (n = 3 per group). j, IFNβ protein levels in culture supernatants from indicated B16-OVA tumour cells treated with IFNγ for 24 h (n = 3 per group). k, Relative levels of IFN-responsive genes (versus non-treated control cells) in indicated B16-OVA tumour cells treated with IFNγ for 24 h (n = 3 per group). l, Relative Ifnb1 and Ccl5 levels (versus non-treated control cells) in control, VDAC2-deficient, caspase-3-deficient, caspase-7-deficient, VDAC2 and caspase-3 co-deficient, or VDAC2 and caspase-7 co-deficient B16-OVA cells treated with or without IFNγ for 24 h (n = 3 per group). m, Relative Ifnb1 and Ccl5 levels in control and VDAC2-deficient B16-OVA tumour cells treated with IFNγ (10 ng ml–1) plus indicated pan-caspase inhibitors (or vehicle) for 24 h. Vehicle label indicates no IFNγ treatment (n = 3 per group). n, Bubble plots showing upregulated (red; Up) and downregulated (blue; Down) pathways in IFNγ-treated VDAC2 and APAF-1 co-deficient B16-OVA tumour cells versus IFNγ-treated VDAC2-deficient B16-OVA tumour cells (n = 3 per group). o, C57BL/6 mice were inoculated with control (n = 8), VDAC2-deficient (n = 8), APAF-1-deficient (n = 7), caspase-9-deficient (n = 7), VDAC2 and APAF-1 co-deficient (n = 8) or VDAC2 and caspase-9 co-deficient (n = 8) B16-OVA tumour cells. Tumour growth (left) and mouse survival (right) were monitored. p, C57BL/6 mice were inoculated with control and VDAC2-deficient B16-OVA tumour cells, followed by vehicle or emricasan (i.p. 20 mg kg–1, twice a day) treatment on days 8–10 after tumour inoculation (n = 10 per group). Tumour growth (left) and mouse survival (right) were monitored. q, Numbers of CD45+ cells (left) and CD8+ T cells (right) in control, VDAC2-deficient, BAK-deficient or VDAC2 and BAK co-deficient B16-OVA tumours on day 14 after tumour inoculation (n = 8 per group). r, Numbers of IFNγ+TNF+ (left) or GZMB+ (right) CD8+ T cells in tumours described in q (n = 8 per group). s, t, C57BL/6 mice were inoculated with control (n = 7), VDAC2-deficient (n = 8), BAK-deficient (n = 7), or VDAC2 and BAK co-deficient (n = 8) B16-OVA tumour cells in combination with isotype (left) or anti-PD-L1 (right) treatment on days 7, 10, and 13 after tumour inoculation. Tumour growth (s) and mouse survival (t) were monitored. Data are representative of two (a–m, o, q–t) or one (p) independent experiments and are mean ± s.e.m. One-way ANOVA (b, c, f, g, i − l, q, r). Two-way ANOVA (d, e, m; tumour size of o, p, s). Mantel−Cox test (survival of o, p, t).
