Extended Data Fig. 5: Characterization of PCC1-induced apoptosis of senescent cells.
From: The flavonoid procyanidin C1 has senotherapeutic activity and increases lifespan in mice
(a) Survival assays of PSC27 cells infected with lentivirus encoding shRNAs against indicated factors. Cells were exposed to PCC1 (100 μM) 7 days after BLEO-caused senescence, for a 3-d period to induce apoptosis. SCR, scramble. P values were calculated by one-way ANOVA with Tukey’s multiple-comparison test. (b) Immunoblot profiling Noxa expression in PSC27 cells infected with lentivirus encoding shRNAs specific for Noxa before treatment with different agents. (c) Immunoblot profiling Puma expression in PSC27 cells, in a workflow resembling that of Noxa. (d) Staining of ROS in senescent PSC27 cells with 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell permeable fluorescent probe indicative of changes in redox state. Signals were measured in a 4-d time course starting from PCC treatment. Scale bar, 20 μm. (e) Examination of ROS by DCFH-DA-staining. Cells were exposed to different agents 7 days after BLEO challenge, for a 3-day period. Staining with DCFH-DA was performed 1 day after addition of individual agents. Scale bar, 20 μm. (f) Statistical comparison of ROS signal intensity measured for cells described in (e). (g) Apoptotic assay of PSC27 cells exposed to in vitro treatments described in (e) by examination of caspase 3/7 activity. Signals from DMSO treatment as a baseline, with indicated P values derived from comparison against PCC1-treated samples. Data are shown as mean ± SD and derive from 3 biological replicates (n = 3 independent assays). P values were calculated by two-sided t-test. (h) Immunoblot of cytochrome c in cells exposed to PCC1. (i) Immunoblot of cytochrome c in cells exposed to PCB2. For (h) and (i), distribution of cytochrome c between mitochondria and cytoplasm was profiled by isolating mitochondria from cytosol supernatants at specialized timepoints (day 0-3 for h, and 3 for i) after procyanidin treatment. COX IV, mitochondrial marker. Data of b-d, h-i are representative of 3 independent experiments. Data of bar graphs (a and f) are shown as mean ± SD and represent 3 biological replicates. ^, P > 0.05. *, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001.
