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. 2020 Feb 21;11:123. doi: 10.3389/fphar.2020.00123

Figure 4.

Figure 4

Antagonistic effects of tempol on the pro-apoptotic action of ATO (2 μM)/AA (2 mM) combination in SW620 cells. (A) Effects of ATO, AA, and ATO/AA combination on the levels of intracellular reactive oxygen species (ROS) as measured by flow cytometry using an oxidation-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Note that ATO/AA combination caused the greatest ROS production as indicated by the highest cell count of DCFH-DA staining. (B) Inhibitory effect of an ROS scavenger tempol (1 mM) on the synergistic enhancement of ROS production induced by ATO (2 μM)/AA (2 mM) combination treatment of SW620 cells, as determined by a ROS fluorescent probe. (a) Control, (b) ATO (2 μM); (c) AA (1 mM); (d) AA (1 mM) + ATO (2 μM); (e) AA (2 mM); (f) AA (2 mM) + ATO (2 μM); (g) AA (2 mM) + ATO (2 μM) + tempol (1 mM). Magnification: ×200. Scale bar: 10 μm. (C) Inhibitory effect of tempol (1 mM) on the synergistic suppression of cell viability induced by ATO (2 μM)/AA (2 mM) combination treatment of SW620 cells. SW620 cells were pretreated with tempol for 1 h before incubating with ATO (2 μM) + AA (2 mM) for 24 h. The data are represented as mean ± SEM (n = 3). ***P < 0.001 vs. Ctl. ### P < 0.001 vs. AA + ATO. (D) Alleviating effects of tempol on the upregulation of Bax protein and downregulation of Bcl-2 protein induced by ATO (2 μM)/AA (2 mM) combination treatment of SW620 cells. (E) Reversing effect of tempol on upregulation of cleaved-caspase-3 levels induced by ATO (2 μM)/AA (2 mM) combination treatment. Beta-actin was used as an internal control. **P < 0.01, vs. Ctl. # P < 0.05 vs. AA and ATO. ## P < 0.01 vs. AA +ATO; n = 5.