TRIP-Br-overexpressing-NIH3T3 fibroblasts proliferate in the absence of mitogenic stimulation as a result of deregulation of the RB/E2F/DP1 transcriptional pathway. V: Vector-only clones, R1: TRIP-Br1-HA-overexpressing clones, R2: TRIP-Br2-HA-overexpressing clones. β-tubulin was used as a loading control. (A) NIH3T3 fibroblasts were transfected with pCDNA3.1 vector (NIH3T3vector-only), pCDNA3.1-TRIP-Br1-HA (NIH3T3TRIP-Br1-HA) or pCDNA3.1-TRIP-Br2-HA (NIH3T3TRIP-Br2-HA), selected by G418, and analyzed by immunoblotting using an anti-HA antibody. Data was obtained from three independent experiments that were performed in triplicates. (B) NIH3T3vector-only, NIH3T3TRIP-Br1-HA and NIH3T3TRIP-Br2-HA fibroblasts were cultured in 96-well plates in DMEM supplemented with 0.2% FBS and were maintained for 72 h at 37°C in a 5% CO2 environment. BrdU incorporation was monitored using a cell proliferation/colorimetric ELISA assay. The fold increase in BrdU incorporation of all clones was calculated relative to that of V16, which was set arbitrarily to 1.0. The error bars represent the standard deviations of three independent experiments performed in triplicates. A Student's t-test was performed and the respective p-values were indicated in the bar chart. (C) Upon serum deprivation, S phase cell counts were significantly higher in the TRIP-Br-overexpressing NIH3T3 clones than the vector-only control. The results shown represent the mean ± SD for each independent R1 (-19 and -20) and R2 clone (-4, 14, 43), compared to all V clones combined (-16, -17, -19), and incorporate data from 3 independent experiments performed in triplicate. A Student's t-test was performed; *indicates p-value < 0.001; **indicates p-value < 0.01 for the comparison of NIH3T3vector-only and NIH3T3TRIP-Br1-HA or NIH3T3TRIP-Br2-HA cells. (D) Upper panel: Semi-quantitative RT-PCR analyses revealed up-regulation of CYCLIN E (CCNE), CYCLIN A2 (CCNA2), CDC6 and DHFR in serum-deprived NIH3T3TRIP-Br1-HA and NIH3T3TRIP-Br2-HA fibroblasts. TS: Thymidylate synthase; 18srRNA was used as a loading control. Data was obtained from three independent experiments that were performed in triplicates. Lower panel: Western blot analyses showed an increase in cyclin E in serum-deprived NIH3T3TRIP-Br1-HA and NIH3T3TRIP-Br2-HA fibroblasts when these cells were immunostained with anti-cyclin E antibodies. Data was obtained from three independent experiments that were performed in triplicates.