NRF2 activity during osteocytogenesis. (A) Undifferentiated IDG-SW3 cells after 3, 7, and 14 days of differentiation were analyzed for NRF2 and KEAP1 protein expression. Tubulin levels were used as a protein load control. (B) NRF2 and KEAP1 levels were quantified and normalized by tubulin levels. Results are plotted as mean ± SEM (n = 3). (C) mRNA expression of Keap1, Nfe2l2, and target genes in undifferentiated IDG-SW3 after 3, 7, and 14 days of differentiation. mRNA levels were quantified by qRT-PCR, normalized by Tbp, and plotted as expression relative to undifferentiated IDG-SW3 (mean ± SEM; n = 8) (D) mRNA expression of Keap1, Nfe2l2, and target genes in primary cultures of osteoblasts and osteocytes. mRNA expression was quantified by RT-qPCR and normalized to Tbp expression. Results are plotted as 2-ΔΔCt (mean ± SEM; n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 using Student's t-test.