ETO enhances Bcl-6–mediated repression of artificial and endogenous target genes. (A) 293T cells were transfected with a reporter construct containing 4 Bcl-6 binding sites upstream of a thymidine kinase promoter driving luciferase gene expression (B6BS-tk-luc) and with vector control (lane 1), Bcl-6 alone (lane 2), Bcl-6 plus the ETO vector control (lane 3), and Bcl-6 plus ETO (lane 4). Transcriptional output was normalized to an internal control (renilla), and multiple repeats were performed to determine standard deviations. P = .01, representing a significant difference between lanes 3 and 4. This experiment was repeated 6 times. (B) 293T cells were transfected with a GFP-expressing plasmid plus the empty Bcl-6 vector (lane 1), Bcl-6 plus the ETO empty vector (lane 2), and Bcl-6 plus ETO (lane 3). After 24 hours of transfection, the GFP-positive cells were sorted using FACS, and the RNA was submitted to RT-PCR for the cyclin D2 and the actin transcript. After 30 cycles, the PCR products were run on a 2% agarose gel, and the intensity of the bands was analyzed with National Institutes of Health (NIH) image software. The intensities of the respective cyclin D2–actin ratios were plotted on a graph (right panel). (C) 293T cells were transfected with a GFP-expressing plasmid plus the empty vector (lane 1), ETO plus the Bcl-6 empty vector (lane 2), Bcl-6 plus the ETO empty vector (lane 3), and Bcl-6 plus ETO (lane 4). After 12 hours of transfection, the GFP-positive cells were sorted using FACS, and the RNA was submitted to reverse transcription followed by real-time PCR for cyclin D2 and GAPDH transcript. This experiment was performed in triplicate to determine standard deviations. Primers used here were first analyzed for their efficiency, which was almost 100%. (D) 293T cells were cotransfected with ETO and Bcl-6. Chromatin immunoprecipitations were performed after 24 hours with rabbit N-terminal ETO antibody, rabbit polyclonal Bcl-6 antibody, or a rabbit IgG control. ChIP products were PCR amplified using primers surrounding the Bcl-6 binding site on the cyclin D2 promoter. NRS and H2O controls are also shown. Error bars represent SD of 6 experiments.
