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. 2020 Feb 19;9(5):636–650. doi: 10.1002/sctm.19-0372

Figure 4.

Figure 4

Inhibition of mitochondrial reactive oxygen species (mtROS) reduces the DSBs and reverses the proliferation block in GARP−/lowASCs. A, ASCs were transduced with LV‐CTRL, LV#18, and LV#19 and incubated with DCFDA (20 μM) and analyzed by flow cytometry. Representative dot plots showing the percentage of DCFDA+ cells in relation to nonstained cells (left panel). Comparison of DCFDA+ NT, LV‐CTRL, LV#18, and LV#19 ASCs (right panel). Data are shown as mean (SD) of three independent experiments. B, NT, LV‐CTRL, LV#18, and LV#19 ASCs were cultured with (white bars) or without 1 mM NAC (black bars), and their proliferation was analyzed using the xCelligence real‐time cell analyzer system. Proliferation is represented as cell index at 160 hours/cell index at 10 hours. Data are shown as mean (SD) of three independent experiments. *P < .05 vs LV#19 control. C, In order to measure mtROS, NT, LV‐CTRL, LV#18, and LV#19 ASCs were cultured in the absence or presence of mitoTEMPO (25 μM), labeled with MitoSOX (20 μM) and analyzed by flow cytometry. Representative dot plots showing the percentage of MitoSOX+ cells in relation to nonstained cells (left panels). Graph showing the percentages of MitoSOX+ NT, LV‐CTRL, LV#18, and LV#19 ASCs (right panel). Data are shown as mean (SD) of three independent experiments. **P < .01 vs LV‐CTRL. D, NT, LV‐CTRL, LV#18, and LV#19 ASCs were cultured without (control, blue bars) or with 25 μM mitoTEMPO (green bars) and the levels of γ‐H2AX were analyzed 4 days after transduction by flow cytometry. Data are shown as mean (SD) or four independent experiments. **P < .01. E, Proliferation of NT, LV‐CTRL, LV#18, and LV#19 ASCs cultured without (blue bars) and with mitoTEMPO (green bars) and apocynin (white bars). Proliferation is shown as cell index at 160 hours/cell index at 10 hours. Data are shown as mean (SD) of three independent experiments. ***P < .001 vs LV#19 control. F, Pearson correlation between the levels of mtROS (MitoSOX+ cells) in NT, LV‐CTRL, and LV#18 and LV#19 ASCs (treated or not with mitoTEMPO) and their proliferative capacity. Pearson r = −.85. Data are plotted from four independent experiments. ASCs, adipose‐derived mesenchymal stromal cells; DSBs, double‐strand DNA breaks; GARP, glycoprotein A repetitions predominant; LVs, lentiviral vectors