Fig. 4

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: The histogram shows the percentage of (C) cycling (Ki67 + ; β-galactosidase-), (Q) quiescent (Ki67-; β-galactosidase-), (St) stressed (Ki67 + ; β-galactosidase +), and (S) senescent (Ki67-; β-galactosidase +) cells three (3DD) and five (5DD) days following treatment with H₂O₂ (iSEN). Panel C: The histograms show the percentage of cycling, quiescent, stressed, and senescent cells in MSCs cultures that were incubated with terpenes for two days, and previously treated with H₂O₂ to induce senescence (iSEN). As a reference, the histograms also show the same cell populations in healthy MSCs cultures (NT) treated with terpenes for two days. The symbols **p < 0.01 and *p < 0.05 indicate statistical significance between the iSEN samples and those treated with terpenes. The symbols ## p < 0.01 and #p < 0.05 indicate statistical significance between healthy MSCs samples and those treated with terpenes. CAR: Carvacrol; EUG: Eugenol; LYC: Lycopene; THY: Thymol. (n = 3 biological replicates ± SD). Panel D: 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 < 0.001 indicate the statistical difference between the iSEN samples and those 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 E: The histogram shows the expression levels of proteins involved in the senescence process. Data were obtained by western blot analysis carried out on MSCs cultures that were incubated with terpenes for two days, 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). Panel F: Western blot analysis of SRC phosphorylation carried out on MSCs cultures, which were incubated with terpenes for two days, 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). Panel G: Representative images of cells stained with anti‐ γH2AX. Cell nuclei were stained with DAPI. The graph shows the degree of ‐γH2AX foci per cell (***p < 0.001 represents statistics significance between iSEN, chosen as reference, and iSEN samples treated with terpenes). (n = 3 biological replicates ± SD)