Figure 3. Nrf2 activation is required for GRh3-induced endometrial cell protection against OGDR. Stable T-HESC cells, with Nrf2 shRNA (“-a/-b”, different sequences) (A–E), the CRISPR-Cas9-Nrf2-KO construct (“ko-Nrf2”) (A–E) or the Nrf2 S40T mutant construct (“Nrf2-S40T”) (F–J), were treated with GRh3 (10 μM) for 8h; Expression of listed proteins in total cell lysates was shown (A and F). Cells were pretreated with GRh3 (10 μM) for 2h, followed by OGD (4h)-reoxygenation (“OGDR”) for applied time periods, then the mitochondrial depolarization (JC-1 green fluorescence, B and G) and superoxide contents (C and H) were tested, with cell viability and necrosis examined by CCK-8 (D and I) and LDH release (E and J) assays, respectively. “Pare” stands for the parental control cells (A–E). “Vector” stands for control cells with empty vector (F–J). Expression of the listed proteins was quantified, after normalizing to the loading control protein (A and F). Error bars stand for mean ± standard deviation (SD, n=5). #p<0.05. Each experiment was repeated three times with similar results obtained. Bar=100 μm (B and G).