FIG. 3.

Effects of ATF3 on Nrf2 in vitro expression. (A) Bar graph showing quantitative real-time PCR (qRT-PCR) results for Nrf2 gene expression in Beas-2b cells in response to cyclic stretch with or without co-exposure to LPS in the presence or absence of ATF3. Absence of ATF3 enhances Nrf2 de novo gene synthesis, while overexpression attenuates Nrf2 de novo gene synthesis. One-way ANOVA within groups (n=2 independent experiment three replicates per group, treatment, and genotype analysis performed separately). *p<0.05 for the comparison between control and LPS treated; and #p<0.05 for the comparison between sh-RNA versus Ad-shATF3 and Ad-βGal versus Ad-ATF3. (B) Western blots showing decreased Nrf2 and increased Keap-1 in response to 24 h treatment with LPS (1 μg/ml) in WT and ATF3−/− BMMs; and (C) increase in Nrf2 and decrease in Keap-1 expression in WT BMMs infected with an adenovirus vector overexpressing ATF3 (Ad- ATF3) compared with the control virus (Ad-β-Galactosidase, βGal). Overexpression of ATF3 in ATF3−/− BMMs results in increased Nrf2 and prevents significant increase in Keap-1 protein expression (n=3). (D) Representative Western blots showing Nrf2 and Keap-1 protein expression in Beas-2b cells with ATF3 silencing and overexpression. ATF3 gene silencing leads to increased Keap-1, while overexpression leads to decreased Keap-1 protein expression in Beas-2b cells exposed to LPS (n=3). Protein expression ratios were normalized to GAPDH. (E) Effect of ATF3 on Nrf2 protein degradation. Cell lysates immunoprecipitated with anti-ubiquitin (IP: Ubiquitin) antibodies and then immunoblotted with anti-Nrf2 antibodies (IB: Nrf2) shows that ATF3 gene silencing (shATF3) results in increased ubiquitination of Nrf2.