Extended Data Fig. 4: Heterochromatin re-formation induced by restoration of SAM reduces susceptibility to genotoxic stress and cell death of old MuSCs.
From: Depletion of SAM leading to loss of heterochromatin drives muscle stem cell ageing
a, Quantification of RFUs of intracellular SAM (n = 4). b, Quantification of RFUs of intracellular putrescine. Freshly isolated old MuSCs were treated with vehicle, putrescine, or ornithine for 48 hr (n = 4). c, After treating old MuSCs as described in (b), doxorubicin was added to the media, and the cells were cultured for another day. (Left) Representative confocal images of γ-H2AX foci. (Scale bar, 5 μm) (Right) The ratio of γ-H2AX foci per cell (n = 4). d, Old MuSCs were treated as described in (c). The ratio of apoptotic cells over total cells was quantified by TUNEL assay (n = 4). e, Quantification of mean RFUs of intracellular spermidine (n = 4). f, Quantification of intracellular SAM measured by SAM ELISA (n = 4). g, Representative western blots of SRM and α-tubulin in old MuSCs (n = 3). h, Band intensity of SRM was normalized to band intensity of α-tubulin (n = 3). i-j, Old MuSCs transfected with siControl or siSrm were stained with antibodies against SAM (i) or spermidine (j). Mean RFUs of intracellular SAM or spermidine in each group of cells were quantified (n = 3 for SAM staining, n = 4 for spermidine staining). k, Old MuSCs were transfected with siControl or siSrm and treated with vehicle, UNC0642, or Chaetocin for 24 hr as indicated. (Left) Representative confocal immunofluorescence images of γ-H2AX foci (Scale bar, 5 μm). (Right) Quantification of the number of γ-H2AX foci per cell (n = 4). l, Quantification of the ratio of TUNEL positive cells over total cells (n = 4). m-n, Freshly isolated human MuSCs were treated as indicated for 48 hr. RFUs of H3K9me3 (m) or HP1α (n) were normalized to RFUs of total H3 (n = 3). o, Human MuSCs treated as described in (m-n) were additionally treated with doxorubicin for 24 hr. (Left) Representative confocal images (Scale bar, 5 μm). (Right) Quantification of the number of γ-H2AX foci per cell (n = 3). p, The human MuSCs treated as described in (o) were subjected to TUNEL assay. The ratio of TUNEL positive cells over total cells was quantified (n = 3). Data are shown as mean ± SD (a-d, f, h-l, o, p) and as median and quartiles (e, m, n). P values were calculated by two-sided unpaired Student’s t-tests (a-f, h-p). *P < 0.05; **P < 0.01; ***P < 0.001.
