Figure 11.

CHOP activates the Puma promoter during ER-stress-induced apoptosis. A, Neurons were cotransfected with the pGL3b or Puma–LUC reporter constructs and either pcDNA3 or pcDNA3–CHOP, and luciferase activity was measured after 24 h. Luciferase activity is reported as the ratio of luciferase activity produced in cells transfected with pcDNA3–CHOP relative to empty pcDNA3 vector for each reporter construct (n = 4; *p < 0.05). B, Cortical neurons derived from Puma^+/+ and Puma^−/− littermates were nucleofected with pGFP and either pcDNA3 [empty vector (EV)] or pcDNA3–CHOP. Neurons were Hoechst stained 48 h after transfection, and the fraction of GFP-positive neurons exhibiting an apoptotic nuclear morphology was determined (n = 3; *p < 0.05). C, Cortical neurons were treated with camptothecin (10 μm; CPT), tunicamycin (3 μg/ml; TM), or thapsigargin (1 μm; TG), and CHOP binding to the Puma promoter was assessed after 12 h by ChIP assay. The level of CHOP binding was quantified by real-time PCR and is reported as fold enrichment over untreated controls (n = 4; *p < 0.05).