Figure 5.

P-AO-treated cells irreversibly stop to self-renew, enlarge and accumulate ROS. (A) Cell cycle progression of the synchronized eMSCs after 24h-P-AO treatment (with Tempol) and subsequent washing. Quantification of the G₀/G₁, S and G₂/M cell fractions in P-AO-treated eMSCs reveals S-G₂/M-block of cell proliferation. (B) Cell cycle distributions of the control and P-AO-treated eMSCs after 24-hour treatment, washing and three day cultivation (96 hours after treatment or 72 hours after washing): G₂/M-blocking after Tempol, resveratrol, NAC and DPI applications (flow cytometry studies). (C) Growth curves after 24h-P-AO-treatment with Tempol, resveratrol or DPI and subsequent washing show irreversibility of cell proliferation block. (D) Flow cytometry analysis of the mean cell fluorescence in the P-AO-treated cells incubated with AOs for 6 hours (left panel) or P-AO-treated cells incubated with AOs for 24 hours, washed and cultured for three more days (96 hours after AO treatment or 72 hours after washing, right panel) and then stained with H₂DCFDA, ROS- sensitive dye. Measurements do not reveal the pro-oxidatve effect of AOs after 6-hour incubation, however, they show a significant increase in ROS level 4 days post AO treatment. (E) Flow cytometry estimation of geometric cell sizes by the means of forward scattering signal (FSC) in the P-AO-treated cells incubated with AOs for 24 hours, washed and cultured for three more days (96 hours after AO treatment or 72 hours after washing, right panel). Results illustrate significant enlargement of the cells 4 days after AO treatment. All data are shown as an average of at least three independent experiments ± SD. *p < 0.05, ANOVA test. Abbreviations: ROS, reactive oxygen species; eMSCs, endometrial mesenchymal stem cells; AOs, antioxidants, namely Tempol (Tem, 2 mM), resveratrol (Res, 60 μM), Diphenyleneiodonium (DPI, 2 μM); P-AO-treated cells, eMSCs exposed to antioxidants at 14 h post-serum stimulation; SD, standard deviation.