Fig. 4
Obesogenic stimuli induce senescence and increase TRF1 levels in ADSC. (A) Quantitative up-regulation of SA-β-gal activity accessed by flow cytometry after 10 or 18 days showed (left) MFI and (right) percentage of cells in the PO group compared to control and PE. (B) (Left: scale bar 50 μm) Representative images of SA-β-gal activity measured by fluorescence microscopy in cultured cells after 10 days of treatment. Nuclei were counter-stained with Hoechst 33342. White dashed lines were manually drawn and represented the contour of cellular membranes. (Right) Fluorescence was quantified as arbitrary units/cell (n = 20–25 cells/treatment in triplicate). (C) (Left) Representative plots from flow cytometry analyzing SA-β-gal activity and cell size (estimated by forward scatter – FSC). (Right) Quantitative analyses (%) of Q2 and Q3 populations in 10 and 18 days of treatment. (D) (Left) TRF1 protein expression (MFI) and (right) percentage (%) of cells were augmented in ADSC treated with PO after 10 days. Dashed lines represent ADSC incubated with 300 μM of hydrogen peroxide (H2O2) for 3 h two days before experiments, treated as a positive control of senescent cells. (E) TRF1 expression and SA-β-gal activity were positively correlated after 10 days of treatment (r = 0.8408, p < 0.0001). (F) CDKN2A (p16INK4A) mRNA expression (relative to RPLP0 housekeeping gene) was quantified after 10 or 18 days of treatment. Data are presented as mean and standard deviation (SD). Differences were considered when p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****), evaluated by one-way ANOVA test followed by Tukey post-test, with a confidence interval of 95%. Abbreviations: CDKN2A: Cyclin-dependent kinase inhibitor 2 A; FCS: forward scatter; PE: pool of plasma from eutrophic individuals supplemented in culture medium; PO: pool of plasma from individuals with obesity supplemented in culture medium; SA-β-gal: Senescence-associated beta-galactosidase; TRF1: Telomeric repeat factor 1