Fig. 5
Biological effects of terpenes on senescent cell cultures. Panel A: Experimental procedure devised to analyze the effects of terpenes on MSCs treated with H2O2(induced senescence—iSEN). Panel B: Flow cytometry chart of Annexin V assay on healthy MSCs (NT), H2O2-treated MSCs (iSEN), and iSEN treated with terpenes. The percentage of apoptotic cells is specified in the histogram. The symbol ****p < 0.0001 indicates statistical significance between the iSEN samples and those treated with terpenes. (n = 3 biological replicates ± SD). Panel C: Western blot analysis of apoptotic pathways performed on MSCs cultures, which were incubated with terpenes for 24 h, and previously treated with H2O2 to induce 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). Supplementary file 2 shows the loading controls. 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 with/without terpenes. 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. Panel E: The histogram shows the percentage of senescent cells undergoing apoptosis (β-galactosidase + , ANXAV +) in H2O2 treated cultures (iSEN), which were incubated with terpenes for 24 h. The symbol ****p < 0.0001 indicates statistical significance between the iSEN samples and those treated with terpenes. (n = 3 biological replicates ± SD). Panel F: Cell cycle plots of healthy MSCs (NT), H2O2-treated MSCs (iSEN), and iSEN treated with terpenes. For every cell cycle phase, the symbols *p < 0.05 and **p < 0.01 indicate the statistical difference between the iSEN samples and iSEN treated with terpenes. The symbols ## p < 0.01 and #p < 0.05 indicate statistical significance between NT and iSEN samples. (n = 3 biological replicates ± SD). Panel G: Western blot analysis of SRC phosphorylation carried out on MSCs cultures, which were incubated with terpenes for 24 h, and previously treated with H2O2 to induce 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).). Supplementary file 2 shows the loading controls. Panel H: The histogram shows the percentage of live cells in H2O2 treated cultures (iSEN), 24 h post-treatment with terpenes. The symbol ****p < 0.0001 indicates statistical significance between the untreated samples and those incubated with terpenes. As a reference, the histograms also show the number of live cells in healthy MSCs cultures (NT). CAR: Carvacrol; EUG: Eugenol; LYC: Lycopene; THY: Thymol. (n = 3 biological replicates ± SD)