Figure 2.

Nuclear factor erythroid 2-related factor (NRF2) deficiency increases MYC transcription and protein expression. Control (wild-type (WT)) and NRF2-deficient (NRF2 knock-out (KO)) OCPs were stimulated with RANKL (50 ng/mL) for the indicated time points. (A) Immunoblot of nuclear protein lysates using NRF2, c-Myc, and Lamin B antibodies. Lamin B served as the loading control. Data are representative of three experiments. (B) The mRNA expression of Myc (relative to the Hprt housekeeping gene) after 6 h of RANKL stimulation (n = 3). (C) Immunoblot of nuclear protein lysates using phosphorylated c-Myc (S62), c-Myc, and Lamin B antibodies. Lamin B served as the loading control. Data are representative of three experiments. (D) Schematic diagram showing the primers (indicated by the black arrows) designed to detect un-spliced, premature Myc mRNA (pre-Myc). (E) Expression of pre-Myc after 6 h of RANKL stimulation (n = 6). (F,G) OCPs were stimulated with RANKL for 6 h and then treated with actinomycin D (10 μg/mL) for 0.25, 0.5, 1, and 3 h. (F) Percent expression of Myc after indicated h after actinomycin D treatment (n = 3). (G) Half-life of Myc transcript in WT and NRF2-deficient OCPs (n = 3). (H) Immunoblot of total cell protein lysates using p-ERK1/2, ERK1/2, p-JNK, p38, IκBα, and α-tubulin antibodies. α-tubulin served as the loading control. Data are representative of three experiments. All data are shown as mean ± s.e.m. * p < 0.05 and *** p < 0.001 using two-way ANOVA in (B,E); NS, not significant using two-tailed, unpaired t-test in (G).