Research Paper Volume 10, Issue 7 pp 1666—1681

Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2

Figure 1. H2S donor treatment is associated with partial rescue from cellular senescence phenotypes. Levels of the senescence-associated total CDKN2A gene expression (A) and levels its alternatively-expressed isoforms p14 and p16 (B) were assessed by qRTPCR in senescent endothelial cells after 24h treatment with H2S donors (Na-GYY4137 at 100 µg/ml, AP39, AP123, RT01 at 10 ng/ml). Data are expressed relative to stable endogenous control genes GUSB, IDH3B and PPIA, and are given normalised to the levels of the individual transcripts as present in vehicle-only treated control cells. Fold change was calculated for in triplicate for three biological replicates. (C). The proportion of cells staining positive for Senescence Associated β-galactosidase (SA-β-Gal) activity following treatment with H2S donors was determined by manually counting the percentage of SA-β gal positive cells. (D) The proportion of cells staining positive for H2Ax marker of DNA damage following treatment with H2S donors was determined by manually counting the percentage of H2Ax positive cells. N = >300 cells for each sample. Statistical significance is indicated by *p<0.05, ** p<0.005, *** p<0.0001 (2 way ANOVA).