Fig. 2. Characterisation of the senolytic potential of PCC1.
a, Measurement of senescent PSC27 cell survival by SA-β-Gal staining. PCC1 was applied at increasing concentrations. P values were calculated by one-way ANOVA with Tukey’s multiple-comparison test. b, Survival of senescent PSC27 cells induced by bleomycin at increasing PCC1 concentrations. c, Apoptotic assay for caspase 3/7 activity. d, Time course survival curves to assess PSC27 cell viability after PCC1 treatment. e, Images of SA-β-Gal staining. TIS, therapy-induced senescence (by bleomycin). Scale bar, 20 μm. Data are representative of three independent experiments. f, Flow cytometry after processing with an annexin V–FITC and PI kit and DAPI staining to determine apoptosis levels. g, Quantification of the percentage of viable (Q4, PI−annexin V−) and apoptotic (Q2 and Q3, PI+annexin V+ and PI−annexin V+, respectively) cells after treatment with vehicle or PCC1 for 3 d (n = 3 biologically independent assays). h, Immunofluorescence staining of PSC27 cells. RS was induced by serial passaging before PCC1 treatment. Red, p16INK4a. Cells at an early passage (P10) were used as a negative control. ABT-263 (1.25 μM) was tested as a positive control. Scale bar, 20 μm. i, Statistics of immunofluorescence staining. j, PCC1-induced senolytic activity after pan-caspase inhibition (20 μM QVD-OPh). k, PD assay of human MSCs. PCC1 was applied on the 8th day after the beginning of experiments as indicated. BLEO, bleomycin. For c,d,k, data are shown as mean ± s.d. and were derived from three biological replicates (n = 3 independent assays). For data in b–d,g,i,j, P values were calculated by two-sided t-tests. In experiments for c–k, PCC1 was used at 100 μM. Unless otherwise indicated, samples were collected for analyses 3 d after PCC1 treatment. Data in bar graphs are shown as mean ± s.d. and are representative of three biological replicates. NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.