Figure 3
The role of an Egr-1 response element in CPZ-induced activation of p21Waf1/Cip1 promoter activity. (A) Promoter reporter assay. C6 cells were transfected with p21-Luc(-150/+38) (wild type; WT) or the Egr-1 mutant construct (mtEgr1). After 48 h of transfection, cells were either left untreated or treated with 20 µM CPZ for 8 h, and then luciferase activities were measured. The firefly luciferase activity was normalized to Renilla activity. Error bars represent the mean ± SD of three independent experiments performed in triplicate. (B) C6 cells were co-transfected with 0.5 µg p21-Luc(-150/+38) and the Egr-1 expression plasmid (pcDNA3.1/Egr1) at increasing concentrations. After 48 h, cells were collected and measured for luciferase activity as in (A). (C) ChIP assay. After C6 cells were treated with 20 µM CPZ for 2 h, chromatins were immunoprecipitated using rabbit anti-Egr-1 antibody (αEgr1), and PCR amplified using primers targeted to the promoter elements (-170 to +42) of the p21Waf1/Cip1 gene. Total genomic DNA obtained from C6 cells was amplified by using the same primers as a control (Input). (D) C6 cells were transiently transfected with 0.5 µg of p21-Luc(-150/+38) plasmid, along with pSilencer/scrambled (Control) or pSilencer/siEgr1 (Egr-1 siRNA), as indicated. The firefly luciferase activity was normalized to the Renilla activity. Error bars represent the mean ± SD of three independent experiments performed in triplicate.
