Fig. 3

CCND1 expression was regulated by EGR1 that directly bound in the promotor of CCND1 in glioma cells. Lysates of U87, U251 and U251SLC cells expressing NT control siRNA, siEGR1 were analyzed by real-time quantitative PCR (a) and by immunoblotting(b). siRNA-mediated knockdown of EGR1 inhibits CCND1 expression. c Chromatin fragments from U251 cells were immunoprecipitated (with antibodies specific to RNA polymerase II (anti- RNA polymerase II; positive control), mouse IgG (IgG, negative control), and EGR1 (anti-EGR1) as indicated. Input, 1% total DNA. After reversal of cross-linking, the immunoprecipitated DNA was amplified by PCR using the specific primers and resolved on 2% agarose gels. ChIP assay demonstrated that EGR1 protein bound to the promoter region of the CCND1 gene. d The immunoprecipitated DNA of U251SLC cells expressing negative control siRNA and siEGR1 was amplified by PCR using the specific primers and resolved on 2% agarose gels. e RT-qPCR analyses the immunoprecipitated DNA of NC-U251SLC and siEGR1-U251SLC. Values were expressed relative to percent input. *P < 0.05, **P < 0.01, and, ***P < 0.001 (mean ± SEM)