Research Paper Volume 12, Issue 11 pp 10370—10380

Keap1-Nrf2 signaling activation by Bardoxolone-methyl ameliorates high glucose-induced oxidative injury in human umbilical vein endothelial cells

Figure 3. Nrf2 silencing or knockout blocks BARD-induced cytoprotection in HG-stimulated HUVECs. The stable HUVECs with Nrf2 shRNA lentiviral particles (“sh-Nrf2”) or the lenti-CRISPR-GFP-Nrf2 knockout (KO) construct (“ko-Nrf2”), as well as the parental control cells (“Pare”), were treated with Bardoxolone Methyl (BARD, at 50 nM) for applied time periods, expression of listed genes was tested by qPCR and Western blotting analyses (A, C, D); The relative ARE activity was examined as well (B); Alternatively, cells were pretreated with BARD (50 nM) for 1h, followed by HG stimulation and cultured for 48h, cell viability (CCK-8 assay, E) and apoptosis (nuclear TUNEL staining assay, F) were tested. Expression of the listed proteins was quantified, normalizing to the indicated loading control protein (A, D). Error bars stand for mean ± standard deviation (SD, n=5). ##p<0.01. vs. “Pare” cells. Each experiment was repeated five times to insure the consistency of experimental results.