Figure 3.

CEBPD regulates CAT expression. (a) Total RNA from U373MG or T98G stable clones was harvested and examined by RT-qPCR to detect CAT expression levels. (b) Left panel shows a schematic representation of the various CAT-based reporter constructs used in this study. Cells were cotransfected with the indicated CAT reporter constructs and siRNAs. After 72 h, cells were lysed for luciferase assay. (c) Left panel shows a schematic representation of the CAT-based reporter mutation constructs. Cells were cotransfected with the indicated CAT reporter mutation constructs and siRNAs. After 72 h, cells were lysed for luciferase assay. (d) CEBPD binds to the CAT promoter. Sheared formaldehyde cross-linked chromatin from U373MG or T98G stable clones was immunoprecipitated with the indicated antibodies and processed for PCR amplification. As a positive control, PCR amplification was also performed with input chromatin that was collected before the IP step. The chromatin was isolated from stable clones. An IP step was performed with IgG or CEBPD antibody. The “-477/-252” and “-188/-5” indicate the PCR products after amplification with specific primers using purified templates from the specific antibody-IP step. (e) Western blot analyses were conducted with the indicated antibodies using protein lysates from U373MG or T98G stable clones. Expression of α-tubulin served as the internal control. Lower panel shows the quantification of CAT protein expression. (f) Cells were harvested from U373MG or T98G stable clones and subjected to catalase activity analysis. Bars represent the means ± SEMs from three independent experiments. Differences among groups were determined with one-way ANOVA followed by Tukey's multiple comparison test. ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05. ns: no significant; shLuc: shRNA for luciferase; shB7, shC7: shRNAs for CEBPD; siNeg: siRNA for negative control; si2895, si2896: siRNAs for CEBPD.