Figure 3. cGAS regulates the senescence-associated secretory phenotype.

(a, b) Conditioned medium (CM) was collected from WT MEFs or cGAS KO MEFs exposed to 40% O₂ for 7 days. Percentages of proliferating cells (WT MEFs) were assessed by BrdU incorporation assay (a) or induction of SA-β-Gal activity was determined by microscopy (b). (c) Cytokine profile of the CM from WT MEFs of cGAS KO MEFs exposed to 40% O₂ for 7 days. Some SASP factors are highlighted in red. (d) WT MEFs, cGAS KO MEFs or STING KO MEFs were incubated in 40% O₂ for 7 days and IL-6 was quantified by ELISA. (e) WI-38 cells were exposed to 40% O₂ treatment for 9 days. On day 7 cells were transfected with a non-targeting control siRNA (si Control) or with siRNAs against cGAS (si cGAS #1, si cGAS #2). Expression of IL-6 was determined by RT-qPCR. (f, g) WT MEFs and cGAS KO MEFs were cultured in the presence of recombinant IFN-β as indicated for 14 days. Cells were stained for SA-β-Gal (f) or cell cycle activity was assessed by EdU incorporation (g). (h, i, j) WT MEFs of IFNAR KO MEFs were cultured under 40% O₂ for 7 days and stained for SA-β-Gal (h), proliferative activity (BrdU incorporation) was assessed by FACS at day 4 (i) or expression of p16^Ink4a was determined by immunoblotting after 7 days (j). Mean and SEM of n=3 (a) or mean and s.d. of n=4 independent experiments are shown (h) or one representative out of 2 independent experiments is shown (c, j). Data shown in (b, d, e, f, g, i) are from n=2 independent experiments with the column representing the mean. P value was calculated by unpaired t-test (h) (ns = not significant). Source data are available in Supplementary Table 1. Unprocessed original blots are shown in Supplementary Fig. 7.