Figure 3

RUNX3 expression in Hep3B cells. CAT (control) and RUNX3 constructs were introduced into Hep3B cells. Polyclonal stably expressing cell lines were selected in the presence of hygromycin. (A) Immunoblot analysis RUNX3 protein expression was analyzed by immunoblot using anti-RUNX3 and anti-His antibodies. Shown here are representative blots from more than three independent experiments. (B) Effect of RUNX3 expression on cell growth Cell proliferative activities were measured by the MTT assay. All results are expressed as ratios to cell number at day 0. Data represent the mean ± S.E. of more than three independent experiments, each with four replicates. *, P < 0.05; **, P < 0.01 (vs. data at 0 h); Student's t-test. (C) Apoptosis determined by DAPI staining Cells were cultured in medium without FBS for the indicated periods. The cells were stained with DAPI, and the percentage of apoptotic cells was examined by fluorescence microscopy (CAT; white bars, RUNX3; black bars). Data represent the mean ± S.E. of more than five independent experiments, each with triplicates. n.s., not significant; **, P < 0.01 (vs. data at 0 h); Student's t-test. (D) Flow cytometry analysis Cells were cultured in medium with or without 10% FBS for 24 h. They were harvested and washed in PBS without Ca²⁺/Mg²⁺, and stained using the Mebcyto™ apoptosis detection kit. The cells were analyzed with a Becton Dickinson FACS Calibur flow cytometer. Shown here are representative plots from more than three independent experiments.