Fig. 2.

cJUN transcriptionally regulates ATF3 expression during the AAR. A: HepG2 cells were transiently transfected with plasmids containing green fluorescent protein (Control), wild-type cJUN (cJUN), or a dominant negative form of cJUN (DN-cJUN). B: in a 2nd series of studies, the cells were transfected with nontarget control shRNA (sh-Control) or cJUN shRNA (sh-cJUN). For A and B, after culture for 48 h, the endogenous ATF3 mRNA content was analyzed by qPCR after the transfected cells were treated with DMEM ± 2 mM HisOH for 6 h. Parallel incubations in DMEM (D) or DMEM + HisOH (H) were used to prepare samples for protein immunoblotting using the primary antibodies indicated. C: HepG2 cells were cotransfected with the pGL3 Firefly luciferase reporter plasmid with no promoter or with the human ATF3 promoter (nt −107/+35) and expression plasmids encoding wild-type (WT) cJUN or a DN-cJUN form. After 48 h, transfected cells were incubated in DMEM ± HisOH for 15 h, and then cell extracts were assayed for luciferase activity and normalized to cell protein content. The data are presented as the averages ± SD for a single experiment containing 3–4 replicates per condition and each experiment was repeated with 2–3 independent batches of cells. CARE, C/EBP-ATF response element. For A–C, *P value of ≤ 0.05 relative to the Control DMEM value, which was set to 1.0, and **P value of ≤ 0.05 relative to the Control HisOH-treated value.