Extended Data Fig. 1. Enhanced cellular senescence in aged lungs and effects of ABT-263 treatment.
A, Validation of the anti-p16 antibodies on transfected cells. HeLa cells were transfected with the control plasmid pcDNA3.1(+) or the plasmid pcDNA3.1(+) encoding hamster p16. Left panel, Cells were labeled with two different anti-p16 antibodies (immunofluorescence). Right panel, Expression of p16 and β actin in HeLa cells expressing or not hamster p16 as assessed by western blotting. B, Left panel, Effect of ABT-263 treatment on the number of p16-positive cells (white arrows) in young and aged lungs as assessed by immunofluorescence. Bars: 25 μm. Right panel, Quantification of p16-expressing cells. The histograms indicate the fold change relative to average intensity in vehicle-treated young animals (n = 8-9). C, SA-β-Gal staining of lung sections from young hamsters and aged hamsters. Increased blue staining (black arrows) indicates a higher number of senescent cells in aged lungs. D, ONPG-based β-Gal activity of lung extracts collected from aged hamsters treated with the vehicle or with ABT-263 (n = 3). E, Left panel, Expression of Bcl-xL and β tubulin in young and aged whole-lung homogenates as assessed by western blotting. Right panel, the relative protein levels normalized to β tubulin are shown (n = 3-6). For all graphs, errors indicate mean ± s.d. Pooled results from two experiments (B) and one of two representative experiments (A, C-E) are shown. Significant differences were determined using the two tailed Mann Whitney U test (D and E) and One-way ANOVA Kruskal-Wallis test (nonparametric), followed by the Dunn’s posttest test (B, right). ** P < 0.01, *** P < 0.001.