Fig. 5: Nrf2 activation is absolutely required for iKeap1-induced osteoblast cytoprotection against H₂O₂.

Stable murine osteoblasts expressing the Nrf2 shRNA (“sh-Nrf2”), a CRISPR/Cas9-Nrf2-KO-GFP-puro construct (“ko-Nrf2”) or scramble control shRNA plus the CRISPR/Cas9 empty vector (“shC+Cas9-C”) were established; Keap1 and Nrf2 mRNA expressions were tested by qRT-PCR assays (A); The osteoblasts were treated with iKeap1 (10 μM) and further cultured for indicated time periods, expressions of listed proteins (B) and mRNAs (C) were tested; Alternatively, the osteoblasts were pretreated with iKeap1 (10 μM) for 2 h, followed by H₂O₂ (400 μM) stimulation, cells were cultured for applied time periods; Cell viability and apoptosis were tested by CCK-8 (D) and TUNEL staining (E) assays, respectively. Stable murine osteoblasts expressing a CRISPR/Cas9-Keap1-KO-GFP-puro construct (“ko-Keap1”) or the CRISPR/Cas9 empty vector (“Cas9-C”) were established; Keap1 and Nrf2 mRNA expressions were tested by qRT-PCR assays (F); The ko-Keap1 osteoblasts were also treated with or without iKeap1 (10 μM) for applied time periods, expressions of listed proteins (G) and mRNAs (H) were tested; Alternatively, the ko-Keap1 osteoblasts were pretreated with iKeap1 (10 μM) s, followed by H₂O₂ (400 μM) stimulation and cells were cultured for applied time periods; Cell viability and apoptosis were tested by CCK-8 (I) and TUNEL staining (J) assays, respectively. Expressions of the listed proteins were quantified and normalized to the loading control. Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. ^#P < 0.05 vs. “shC+Cas9-C”/“Cas9-C” osteoblasts. “n.s.” stands for the non-statistical difference (H–J). Experiments were repeated five times, with similar results obtained.