Fig. 6
Pathways involved in senotherapeutics action of terpenes. Panel A: The histogram depicts the percentage of apoptotic cells (Annexin V +) 24 h following SAHA treatment of MSCs either in the presence or absence of different amounts of Z-VAD-FMOC. NT: not treated cells; DMSO: cells treated with Dimethyl sulfoxide (DMSO), a polar aprotic solvent that dissolves Z-VAD-FMOC. The symbol ****p < 0.0001 indicates statistical significance between the specified samples. Panel B: The histograms show the percentage of apoptotic cells (Annexin V +) in H2O2-treated MSCs (iSEN) 24 h following treatment with terpenes, either in the presence or absence of 25 µM Z-VAD-FMOC. NT: not treated cells. The symbols ****p < 0.0001 and ***p < 0.001 indicate statistical significance between the specified samples. (n = 3 biological replicates ± SD). Panels C: Western blot analysis carried out on MSCs cultures, which were incubated with terpenes in presence or absence of Z-VAD-FMOC for 24 h, and previously treated with H2O2 to induce senescence (iSEN). NT: healthy MSCs. The cleaved caspase (CASP8) appears as smeared band. Panels D: Western blot analysis of AIF carried out on enriched cytoplasmic fraction (C), enriched mitochondrial fraction (M) and enriched nuclear fraction (N) of 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 graph shows the AIF expression levels in the analyzed subcellular fractions. Supplementary file 2 shows the loading controls. Panel E: Intracellular ROS detection by DCF-DA in H2O2-treated MSCs (iSEN) incubated with terpenes for different time points either in the absence (upper plots) or presence (lower plots) of glutathione (GSH). NT: healthy MSCs; CAR: Carvacrol; EUG: Eugenol; LYC: Lycopene; THY: Thymol. Panel F: The histograms show the percentage of apoptotic cells (Annexin V +) in H2O2-treated MSCs (iSEN) 24 h following treatment with terpenes, either in the presence or absence of glutathione (GSH). NT: not treated cells. The symbols ****p < 0.0001 and ***p < 0.001 indicate statistical significance between the specified samples. Representative plots of apoptosis analysis are also reported. (n = 3 biological replicates ± SD)