Early Growth Response 1 (Egr1) Is a Transcriptional Activator of NOX4 in Oxidative Stress of Diabetic Kidney Disease

Figure 2

Changes in NOX4 expression with overexpression and knockdown of Egr1 in TGF-β1-treated HK-2 cells. (a, b, and c) Egr1 and NOX4 protein were measured using Western blotting assays in HK-2 cells treated with TGF-β1 (10 ng/mL) at 0 h, 0.5 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, and 48 h. (d and e) Semiquantitative levels of Egr1 and NOX4 protein. (f) Cells were treated with either pENTER-Egr1 overexpression plasmid or with a pENTER vector for 48 h and then exposed for 3 h and 24 h to TGF-β1 (10 ng/mL). Levels of Egr1 were detected at 3 h, and levels of NOX4 and α-SMA were detected at 24 h after TGF-β1 (10 ng/mL). Levels of Egr1 mRNA were measured by RT-qPCR. (g) Levels of α-SMA mRNA were measured by RT-qPCR. (h and i) Levels of Egr1, NOX4, and α-SMA protein were measured using Western blotting assays. (j) Cells were either silenced with siEgr1 or treated with a scrambled control RNA for 48 h and then exposed for 24 h to TGF-β1 (10 ng/mL). Levels of Egr1 mRNA were measured using RT-qPCR. (k) Levels of α-SMA mRNA were measured using RT-qPCR. (l and m) Levels of Egr1, NOX4, and α-SMA protein were measured using Western blotting assays. The above results are expressed as foldchange over baseline. Values are the mean ± SD. and versus control group. (n) ChIP test to explore Egr1 binding to the NOX4 promoter. ChIP was performed using an anti-Egr1 antibody or a negative control IgG antibody in HK-2 cells treated for 3 h and 24 h with TGF-β1 (10 ng/mL). Immunoprecipitated DNA was subjected to RT-qPCR using specific NOX4 primers that included the Egr1 binding sites. (o) Dual-luciferase reporter gene assay to explore Egr1 binding to the NOX4 promoter.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)