Fig. 5 |. Characterization of autophagy-deficient microglia and cellular senescence in vitro.

a,b, Primary microglia were cultured from Atg7^cKO microglia (n = 8, littermate control n = 9) or Atg14^cKO microglia (n = 9, littermate control n = 9). The number of microglia was counted. P < 0.0001 for both genotypes. c,d, The levels of CDKIs, p21^Cip1, p16^Ink4a and p27^Kip1, were measured by western blot from Atg7^cKO microglia (n = 8 for p21^Cip1 and p16^Ink4a; n = 6 for p27^Kip1) and littermate controls (n = 9 for p21^Cip1 and p16^Ink4a; n = 7 for p27^Kip1) or Atg14^cKO microglia (n = 5, littermate controls n = 5, for all CDKIs). Actin was used as loading controls. For p21^Cip1, P = 0.0009 for Atg7^cKO; P = 0.002 for Atg14^cKO. e, Senescence-associated β-galactosidase activity (SA-β-Gal) was measured from Atg7^cKO microglia (n = 8, littermate controls n = 9) or Atg14^cKO microglia (n = 9, littermate controls n = 9). P = 0.0092 for Atg7^cKO; P = 0.01 for Atg14^cKO. Scale bar, 20 μm. f, Cell sizes were measured from Atg7^cKO microglia (340 cells, n = 3; littermate controls 369 cells, n = 3) or Atg14^cKO microglia (306 cells, n = 6; littermate controls 332 cells, n = 4). P < 0.0001 for genotypes. Scale bar, 20 μm. g, Experimental scheme for SASP analysis. h,i, Venn diagram (h) and heat map (i) showing commonly upregulated (top) or downregulated (bottom) secreted proteins from Atg7^cKO (P < 0.05, FC >1.25 or <0.8) and Atg14^cKO microglia (FDR <0.05, FC >1.25 or <0.8) compared with controls. j, The levels of C3 protein in cell lysate (lanes 1 and 2) and medium (lanes 3 and 4) from Atg7^cKO microglia (n = 10) and littermate controls (n = 9) were measured through western blot and quantified. P = 0.0046. P values were calculated by unpaired two-tailed Student’s t-test (a–e and j) or two-tailed Mann–Whitney U test (f). All values are reported as mean ± s.e.m. Source numerical data and unprocessed blots are available in source data.