Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway
Figure 7
HRG1 (SLC48A1) gene expression and protein analysis in NRF2KD, K562, GM12878, and K562 KEAP1KD cells. (a) Transient silencing of NRF2 in three cell lines (A549, BEAS-2B, and HepG2) reduced expression of HRG1. Most significant HRG1 reduction was observed in HepG2 followed by A549. (b) Chemical activation of NRF2 using SFN (10 μM, 24 hours) induced HRG1 expression in GM12878, but high HRG1 baseline levels remained unchanged in SFN-induced K562 cells. K562 cells treated with 5 hours of 10 μM SFN showed an increase in HRG1 protein indicating a response to chemical activation at the shorter timepoint. (c) Genetic activation of NRF2 by stably silencing its negative regulator KEAP1 induced HRG1 in K562 cells at mRNA and protein level. (d) Two different shRNA clones showed an increase in HRG1 as compared to non-target shRNA control. mRNA values were normalized to BACT or 18S. Bars represent an average of 3–6 independent experiments ± SEM; , . A 30 kDA HRG1 band was confirmed by protein markers, and the membrane was stripped and reprobed with β-actin as a loading control.
