FIGURE 4.

The up-regulation of HO-1, COX2, and NRF2 is indirectly associated with NO-induced cytoprotective effect in hCSCs. A, representative images of Western blot showing the dose-dependent increase in expression levels of HO-1 and COX2 and phosphorylation levels of NRF2 in response to DETA-NO preconditioning. B, representative images of Western blot showing the time-dependent up-regulation of HO-1, COX2, and NRF2 with 250 μm DETA-NO preconditioning (PC). C, representative images of Western blot showing expression levels of HO-1 and COX2 and phosphorylation levels of NRF2 after DETA-NO withdrawal over 24 h. The quantitative data for Western blots shown in A–C are provided in supplemental Fig. 4. D, hCSCs expressing shRNAs of HO-1, COX2, and NRF2 were preconditioned with 250 μm DETA-NO for 12 h and then stimulated with H₂O₂. The cytotoxicity induced by oxidative stress was evaluated by LDH release assay. E, after preconditioning (250 μm DETA-NO for 12 h), hCSCs expressing shRNAs of HO-1, COX2, and NRF2 were recovered (RC) for 24 h by refreshing medium. The cytotoxicity induced by oxidative stress was evaluated by LDH release assay. Data (both D and E) are presented as the mean percentage by comparison with scrambled (Scr) shRNA control with S.D. (means ± S.D.). Error bars represent S.D. ** indicates p < 0.01 versus scrambled shRNA group; n = 3 independent experiments. RC, recovery.