Fig. 4
From: The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35
LncNB1 activates DEPDC1B gene transcription through the transcription factor E2F1. a Wild type and E2F1-binding site deletion mutant (Δ20bp) DEPDC1B gene promoters were cloned into a pGL3 firefly-luciferase (Firefly_LUC) construct. DOX-inducible control shRNA, lncNB1 shRNA-1, and lncNB1 shRNA-2 BE(2)-C cells were transfected with the wild type or Δ20bp deletion mutant DEPDC1B gene promoter pGL3 constructs, followed by treatment with vehicle control or DOX and luciferase assays. Percentage change in luciferase activity, measured as relative light unit (RLU) due to DOX treatment as compared with vehicle control treatment, was normalized by the luciferase activity of DOX-inducible control shRNA cells treated with vehicle control. Data were shown as the mean ± standard deviation, and evaluated by one-way ANOVA. *, **, and *** indicated P < 0.05, 0.01, and 0.001 respectively. b, c BE(2)-C and Kelly cells were transfected with control siRNA, E2F1 siRNA-1, or E2F1 siRNA-2 for 48 h, followed by RT-PCR analysis of DEPDC1B (b) and immunoblot analysis of DEPDC1B, E2F1, and N-Myc (c). Data were shown as the mean ± standard error of three independent experiments, and evaluated by one-way ANOVA. *, **, and *** indicated P < 0.05, 0.01, and 0.001 respectively. d ChIP assays were performed with a specific anti-E2F1 antibody (Ab) in DOX-inducible lncNB1 shRNA-1 or shRNA-2 BE(2)-C cells after treatment with vehicle control or DOX, followed by PCR with primers targeting different regions of the DEPDC1B gene promoter (−600 bp, transcription start site (TSS) or +600 bp). e BE(2)-C cells were co-transfected with pCMV14-empty vector or pCMV14-E2F1 plus pCMV10-3 × Flag-DP1 constructs in combination with wild type or E2F1-binding site (Δ20bp) deletion mutant DEPDC1B gene promoter pGL3 Firefly_LUC construct. In d, e data were shown as the mean ± standard deviation of three independent experiments, and evaluated by two-sided unpaired Student’s t-test. ** and *** indicated P < 0.01 and 0.001, respectively. Source data are provided as a Source Data file
