Fig. 2.

BCAA sensitized ADSCs to detrimental stress-induced death and premature senescence. a a1 panel, representative images of TUNEL staining. ADSCs were incubated with increasing doses of BCAA for 24 h with hydrogen peroxide (100 μM)-induced stress. a2 panel, quantification of TUNEL staining intensity. b Viability of ADSCs as determined by CCK-8 assay. c Top panel, representative blots showing cleaved caspase-3, caspase-3, and β-tubulin levels. Bottom panel, ratio of quantified cleaved caspase-3 expression to caspase-3 expression. d d1, representative images of SA-β-gal stained cells. ADSCs were incubated with the indicated BCAA under hydrogen peroxide (100 μM)-induced premature senescence as methods described. d2, quantification of SA-β-gal-positive cells. e mRNA levels of Il1b and Il6 as measured by RT-qPCR and normalized to the level of Actb mRNA. f Representative western blot images and quantification values of P21 and P16. The expression of β-tubulin was used as the loading control. g Left panel, schematic diagram showing ADSCs exposed to hydrogen peroxide (100 μM) and treated with increasing doses of BCAA for 24 h. Twelve hours after the ADSCs were washed, fresh or conditioned medium was added to NRVMs subjected to normoxia or H/R. Right panel, NRVM viability was determined by CCK-8 assay. The data are shown as the means ± SD. The data were analysed by one-way ANOVA, followed by Bonferroni post hoc test. Actb β-actin, ADSCs adipose tissue-derived mesenchymal stem cells, BCAA branched chain amino acids, CCK-8 Cell Count Kit-8, CM conditioned medium, FM fresh medium, H/R hypoxia/reoxygenation, Il1b interleukin-1β, Il6 interleukin-6, NRVMs neonatal rat ventricular myocytes, SA-β-gal senescence-associated β-galactosidase, TUNEL transferase-mediated dUTP nick-end labeling