Fig. 2

Distinct effects of AICAR, NAM, and concomitant AICAR+NAM treatment on senescence-associated changes of MSCs and total cellular reactive oxygen species (ROS). MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. a Phase-contrast images of MSCs (P10) (scale bar = 500 μm), SA-β-gal expression, visualized using light microscopy (scale bar = 100 μm), and fluorescent micrograph (scale bar = 50 μm) of the Acridine Orange stained MSCs at P10 of the four groups. b Left panel: the surface area of the MSCs (P10), calculated using ImageJ software, indicates that cells treated with AICAR alone or AICAR+NAM displayed a significantly lower cross-sectional surface area compared to the NAM-treated cells and the untreated group. Middle panel: prevalence of the SA-β-gal-positive cells, calculated as the number of blue cells per the total number of cells counted. Our data show that treatment with AICAR and NAM reduces the expression of SA-β-gal. Right panel: prevalence of senescent cells determined by the number of green fluorescence-emitting cells per the total number of cells counted. Untreated cells displayed the highest frequency of cells emitting green fluorescence and the least frequency of red fluorescence-emitting cells, compared to the treatment groups. Each bar indicates mean ± SD. c Total cellular ROS was measured at P5 and P10 by staining with DCFDA, followed by flow cytometry analysis (n = 3 independent experiments). d Comparison between total cellular ROS of the study groups at P10 (n = 3 independent experiments). Each bar indicates mean ± SD (*p < .05; **p < .01; ***p < .001; ****p < .0001). Abbreviations: SA-β-gal, senescence-associated β-galactosidase; ROS, reactive oxygen species; DCFDA, 2′,7′-dichlorofluorescin diacetate; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; NAM, nicotinamide