Figure 4 | Cell Death & Disease

Figure 4

From: NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation

Figure 4

A specific NOX1 inhibitor reduces ROS accumulation and promotes long-term expansion of aASCs. Abdominal ASCs (Abd) were propagated under normoxic oxygen conditions (21% oxygen) in the presence of an NOX1-specific inhibitor ML171 (0, 0.5, 2.5 and 5 μM). The following analyses were performed at the indicated passages (P). (aI) ROS accumulation was evaluated by FACS (fluorescence-activated cell sorting) analysis using DCFDA staining, and the results of untreated aASCs compared with aASCs treated with 5 μM inhibitor are shown. (aII) Starting from P3, an additional population with a low DCFDA signal appeared (marked by an arrow). Dual staining of cells by DCFDA and aqua blue dye (an amine viability dye) was performed and analyzed by FACS. Results demonstrated that the majority of low DCFDA cells were aqua blue-positive dead cells. (aIII) RNA expression level of different antioxidant enzymes was compared between cells that were cultured with NOX1 inhibitor (gray line) or without it (black line). Error bars represent S.D. (bI) The percent of dead cells was evaluated by aqua blue staining. The division rate of untreated aASCs was compared with aASCs that were treated with different concentrations of NOX1 inhibitor (bII), and their long-term propagation of cells was evaluated (bIII). (bIV) The number of cells of untreated aASCs and aASCs treated with 2.5 μM NOX1 inhibitor was compared at the indicated passages in two independent experiments. (bV) Activities of caspase-3/7 in untreated aASCs and aASCs treated with 2.5 μM were measured by a Caspase-Glo 3/7 Assay Kit. Error bars represent S.E.M. (Student’s two-tailed t-test for equal (bII) or unequal (bIV) variance). **P<0.01 and *P<0.05. (c) The RNA expression level of NOX1 and various cytokines was compared between untreated aASCs and aASCs treated with 5 μM NOX1 inhibitor by quantitative reverse transcription-PCR (qRT-PCR) at the indicated passages. Error bars represent S.D. Untreated aASCs (Abd (0)). aASCs treated with 0.5 μM, 2.5 μM and 5 μM NOX1 inhibitor (Abd (0.5), Abd (2.5), Abd (5), respectively)

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