Fig. 4: MitoCa²⁺ promotes autophagy by activating IP3R-mediated Ca²⁺ release from the ER.

a Representative images and quantification of the number of autophagosomes in young ISCs (3 d, green) with indicated genotypes by anti-ATG8a staining (red). Data show mean ± S.E.M (n = 99 and 126 cells, from left to right) based on two-tailed unpaired t-test. ****P  <  0.0001. Scale bars, 5 μm. b Quantification of the number of ZFYVE1, ATG6, ATG9 and ATG13 positive puncta in young ISCs (3 d) with indicated genotypes. Data show mean ± S.E.M (n = 89, 112, 109, 65, 114, 89, 132 and 128 cells, from left to right) based on two-tailed unpaired t-test. ****P  <  0.0001. c Quantification of relative MitoCa²⁺ levels in young ISCs (3 d) with indicated genotypes. Data show mean ± S.E.M (n = 102, 123 and 118 cells, from left to right) based on one-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. d Working scheme: MCU overexpression might activate IP3R (question mark) and cause Ca²⁺ release from ER, forming Ca²⁺ oscillations required for isolation membrane (IM) nucleation and autophagy initiation. e Representative images and quantification of relative ERCa²⁺ levels in young ISCs (3 d, red) with indicated genotypes by ER-GCaMP6 (green). Typical ISC (red) was circled by dashed lines. Data show mean ± S.E.M (n = 140, 115, 113 and 127 cells, from left to right) based on one-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. Scale bars,10 μm. f–h Quantification of the number of acidic lysosomes in young ISCs (3 d) with indicated genotypes, respectively. Data show mean ± S.E.M (n = 95, 82, 90, 94 cells in f, n = 95, 71, 94, 65 cells in g and n = 99, 92 cells in h, from left to right) based on one-way ANOVA with Tukey’s multiple comparison test (f, g) and two-tailed unpaired t-test (h). ****P  <  0.0001. i, j Quantification of the intensity of anti-γH2AvD signals in aged ISCs (30 d) with indicated genotypes, respectively. Data show mean ± S.E.M (n = 13, 9, 10, 11 guts in i and n = 11, 9 guts in j, from left to right) based on one-way ANOVA with Tukey’s multiple comparison test (i) and two-tailed unpaired t-test (j). ****P  <  0.0001. Each experiment was repeated for 3 times (a–c, e–j). See Supplementary Table 1 for genotypes. Source data are provided as a Source Data file.