Confirmation of E2F1 and E2F2 as downstream genes of KDM5B. (A and B) Expression levels of E2F1 (A) and E2F2 (B) in SW780, A549 and SBC5 cells were analyzed by real-time PCR after treatment with siRNAs targeting KDM5B (siKDM5B) and EGFP (control; siEGFP = 1). Relative mRNA expression is an arbitrary value, but for each cell population, expression was normalized to GAPDH and SDH expressions. Statistical analysis was based on nine independent experiments. P-values were calculated using Student's t-test. (C) E2F1 and E2F2 expressions as measured by real-time PCR were significantly up-regulated in bladder tumor tissues compared to non-neoplastic bladder tissues, in proportion to that of KDM5B expression in tumor tissue. We analyzed 13 cancer and normal tissues, and Spearman's rank correlation coefficient was used for the statistical analysis. (D) Knockdown of KDM5B expression repressed the transcriptional activity of E2F. Results are the mean ± SD in three independent experiments. P-values were calculated using Student's t-test. (E) Validation of KDM5B and E2F1 expressions at the protein level. Lysates from A549 and SBC5 cells after both siKDM5B#1 and siKDM5B#2 treatments were immunoblotted with anti-KDM5B, E2F1, E2F2 and actin antibodies. Expression of actin served as an internal control.