Extended Data Fig. 7: Increased autophagy alleviates NCOA7 deficiency-related GC senescence and ovarian aging both in vitro and in vivo.
From: Stress granule clearance mediated by V-ATPase-interacting protein NCOA7 mitigates ovarian aging
(a) Western blot of LC3 and p62 protein levels in WT and NCOA7-KO KGN cells treated with DMSO or rapamycin. (b) SA-β-gal staining of WT, NCOA7-KO, and NCOA7-mutant KGN cells. Scale bar: 50 μm. (c) Immunofluorescence and quantitative analysis of G3BP1 and NCOA7 in WT and NCOA7-KO KGN cells (n = 5 biological replicates per group) treated with DMSO or rapamycin. Scale bar: 20 μm. **P = 0.0013, *P = 0.0173. (d) Immunofluorescence and quantitative analysis of SGs in ATG7-knockdown KGN cells (n = 10 views in quantification of stressed cells; n = 20 views per group in quantification of SGs per cell). Scale bar: 15 μm. ****P < 0.0001, ns P = 0.0738; ****P < 0.0001, ns P = 0.8588. (e) Quantification of cell proliferation of GCs from 3-NP-treated WT and Ncoa7−/− mice treated with DMSO or rapamycin (n = 3 biological replicates per group). *P = 0.0351, *P = 0.0131. (f) Quantification of expression levels of SASP genes (Il1b, Il8, Mmp3 and Ccl2) via RT‒qPCR in GCs isolated from 3-NP-treated WT and Ncoa7−/− mice treated with DMSO or rapamycin (n = 4 biological replicates per group). ns P = 0.8979, ns P = 0.7816, ns P = 0.3738, ns P = 0.6572, *P = 0.0376, *P = 0.0321, **P = 0.0070, **P = 0.0045. (g) Quantification of the ovary index in 3-NP-treated WT and Ncoa7−/− mice treated with DMSO or rapamycin (n = 8 biological replicates per group). ns P = 0.2740, ns P = 0.0695. Data in c-g are expressed as means ± s.e.m, and compared by two-tailed Student’s t-test.
