Extended Data Fig. 7. Senescent microglial BV2 cells display cGAS–STING-dependent innate immune activation.
a–d, BV2 cells were irradiated (10 Gy, IR), then DMSO- or H151-treated (daily, 1 μM, day 4-6 post-irradiation). mRNA expression levels of senescence markers (n = 5 experiments) (a), Western blot characterization (b), and senescence-associated-β-galactosidase staining (n = 1, represents n = 3 experiments) (c). Scale bars, 200 μm. mRNA expression levels of proinflammatory and interferon-stimulated genes (d) in control and irradiated, DMSO- or H-151-treated cells, measured for each experiment (n = 4) relative to irradiated DMSO-treated cells. e, cGAMP levels in cell lysates of control and irradiated BV2 cells (n = 7 experiments). f, mRNA expression levels of proinflammatory and interferon-stimulated genes in control and irradiated WT and cGAS-KO BV2 cells (n = 4 experiments). g, Senescence-associated-β-galactosidase (left, n = 3 FOV) and % EdU+ cells (right) from control (n = 8 FOV) and irradiated (n = 3 FOV) WT BV2, H-151-treated or not as in (a) and cGAS-KO BV2 (n = 3 FOV), represents n = 3 experiments. h, Experimental set-up for the analysis of mtDNA-depleted BV2 cells (ρ0 BV2) (left). Mitochondrial DNA sequence Mito levels in ρ0 BV2 whole cell lysate (middle), and in the cytosol after irradiation (right) (n = 2 experiments). i, mRNA expression levels of proinflammatory and interferon-stimulated genes in ρ0 BV2 cells stimulated with LPS or dsDNA 90mer transfection (n = 3 experiments). j, mRNA expression levels of proinflammatory and interferon-stimulated genes in control and irradiated ρ0 BV2 cells, measured for each experiment (n = 3) relative to irradiated untreated (IR) cells. Data are mean ± s.e.m. P values were obtained with two-sided paired ratio Student’s t-test (a,d,e,j), two-sided unpaired Student’s t-test (f), and one-way ANOVA followed by Tukey’s multiple comparisons tests (g,i). uPAR, urokinase-type plasminogen activator receptor.