Fig. 4: SMARCA4/2 loss inhibits apoptosis by constricting IP3R3-mediated Ca2+ flux. | Nature Communications

Fig. 4: SMARCA4/2 loss inhibits apoptosis by constricting IP3R3-mediated Ca2+ flux.

From: SMARCA4/2 loss inhibits chemotherapy-induced apoptosis by restricting IP3R3-mediated Ca2+ flux to mitochondria

Fig. 4

a Immunoblots of SCCOHT-1 cells ± SMARCA4 (A4) and ITPR3 (R3) perturbations. shR3: shRNA targeting ITPR3. Changes of cytosolic (b) and mitochondrial (c) Ca2+ contents in SCCOHT-1 cells ± A4 and R3 perturbations upon histamine stimulation. b 43 control (Ctrl), 30 A4, 51 A4 ShR3♯1, and 50 A4 ShR3♯2 cells from n = 4 independent experiments were analyzed. c 31 Ctrl, 30 A4, 50 A4 ShR3♯1, and 50 A4 ShR3♯2 cells from n = 4 independent experiments were analyzed. d Immunoblots of H1703 cells ± A4 and R3 perturbations. siR3 siRNA targeting ITPR3. Changes of cytosolic (e) and mitochondrial (f) Ca2+ contents in H1703 cells ± A4 and R3 perturbations upon histamine stimulation. e 64 Ctrl, 70 A4, and 64 A4 siR3 cells from n = 3 independent experiments were analyzed. f 53 Ctrl, 53 A4, and 50 A4 siR3 cells from n = 3 independent experiments were analyzed. g Immunoblots of OVCAR4 cells with ITPR3 knockdown 48 h post cisplatin treatment. cl cleaved. h Annexin V+/PI− apoptotic cell population determined by flow cytometry in OVCAR4 cells described in g. Immunoblots of H1703 cells ± R3 overexpression (i) or ±A4 and R3 perturbations (j). Cells were collected 72 h after the treatment. k Annexin V+/PI− apoptotic cell population determined by flow cytometer in H1703 cells described in j. b, c, e, f Left: traces of cytosolic or mitochondrial Ca2+ contents upon 100 μM histamine stimulation (mean ± SEM). Right: quantification of the maximal Ca2+ signal peaks induced by histamine (mean ± SD). The Ca2+ probes R-GECO (R-GECO F/F0) and CEPIA-2mt (CEPIA-2mt F/F0) were used to monitor cytosolic and mitochondrial Ca2+, respectively. Arb. units arbitrary units. One-way ANOVA followed by Dunnett’s test for multiple comparisons to Ctrl, p values (p): b A4—0.0003, shR3#1—0.6223, shR3#2—0.9866; c A4 < 0.0001, shR3#1—0.9109, shR3#2—0.9845; e A4—0.0038, siR3—0.4232; f A4—0.0007, siR3—0.1620. h, k Mean ± SD, n = 3 independent experiments, one-way ANOVA followed by Dunnett’s test for multiple comparisons, p values (p): all <0.0001. *p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant.

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