Fig. 2
Set up of experimental procedure for senotherapeutics analysis. Panel A: The SRC signaling pathways in apoptosis and senescence according to the study by Anerillas and co-authors (2022). Panel B: The histogram displays the expression levels of pSRC(Y416) and total SRC. The upper histograms present data obtained from western blot analysis conducted on MSCs incubated with SAHA at various time points. The lower histograms illustrate western blot analysis conducted on MSCs incubated with H2O2 at various time points (induced senescence – iSEN). As a reference, the histograms also show the expression levels in healthy MSCs cultures (NT). The symbols ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 indicate statistical significance between the specified samples. On the top of every histogram, representative western blot bands of the analyzed proteins are reported. (n = 3 biological replicates ± SD). Panel C: Flow cytometry chart of Annexin V assay on SAHA treated cells. The histogram depicts the percentage of apoptotic cells (Annexin V +) following SAHA treatment. Data refer to analysis at 2, 4, 8, 18, 24, and 32 h post-treatment. NT: not treated cells. The symbol ****p < 0.0001 indicates statistical significance between the control (NT) and SAHA treated samples. (n = 3 biological replicates ± SD). Panel D: The pictures show representative images of senescent (Ki-67-; β-galactosidase +) and apoptotic (ANXAV +) cells in healthy control samples (NT) and induced senescence (iSEN) samples in presence of ABT-737. Cells were stained to identify nuclei (DAPI in blue), Ki67 (red), Annexin V (ANXAV in green), and to evaluate β-galactosidase activity (dark gray). We used a Leica CTR500 microscope, equipped with a DCF3000G digital monochrome camera. The β-galactosidase activity was detected as a gray stain using this configuration. This method allowed us to identify cells that exhibited a visible light signal β-galactosidase along with others expressing fluorescent signals within the same cell. The arrows indicate cells that are positive for senescence and apoptosis markers. The histogram shows the percentage of senescent cells (iSEN) undergoing apoptosis at 8, 16, 24, and 32 h post-ABT-737 treatment. The symbol ****p < 0.0001 indicates statistical significance between the iSEN samples and those treated with ABT-737. (n = 3 biological replicates ± SD)