Extended Data Fig. 3: ADAR1 downregulation does not induce robust SASP.
a, Immunostaining for macroH2A1.2 in IMR90 cells with or without ADAR1 knockdown (shADAR1 #1). Scale bar = 10 μm. Asterisks indicated inactivated X chromosome in female IMR90 cells and arrows indicate examples of macroH2A1.2 positive SAHF. The experiment was repeated three times independently with similar results. b-c, Immunostaining for macroH2A1.2 in control proliferating and replicative senescent WI38 cells with or without ATG7 knockdown (b). Cells with macroH2A1.2 positive foci were quantified (> 200 cells) (c). Scale bar = 10 μm. d, Global RNA A/I editing was analysed based on the RNA-seq A/G reads in the indicated cells. e, Changes in expression of SASP genes in the indicated ADAR1 knockdown vs. control IMR90 cells (KD/Ctr) determined by RNA-seq analysis. SASP genes with at least 10 counts at least 1 FPKM level are reported. f, Secreted cytokines in conditioned media from IMR90 cells with or without ADAR1 knockdown (shADAR1 #1) was detected using an antibody-based cytokine array. Conditioned media collected from RAS-induced senescent IMR90 cells was used as a positive control. g-h, Expression of the indicated proteins was determined by immunoblot in IMR90 cells expressing shControl or shADAR1s (g) or sgGFP control or sgADAR1 (h). The experiment was repeated three times independently with similar results. i-j, Representative images of SA-β-gal staining of IMR90 cells cultured with conditioned media from indicated cells (i). Percentages of SA-β-gal positive cells were quantified (from > 200 cells) (j). k-m, IMR90 cells expressing shControl or shADAR1 (#1) with or without ectopically expression of wildtype ADAR1 p110 or a deaminase inactive mutant E912A ADAR1 p110 were analysed for SA-β-Gal activity (k) and positive cells were quantified (from > 200 cells) (l). In addition, the indicated cells were subjected to colony formation assay (m). Scale bars = 100 μm. Data represent the mean ± s.d. in l and m, or s.e.m. in c and j of three biologically independent experiments. P values were calculated using a two-tailed Student’s t-test. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.
