Figure 3

REP1 confers resistance to serum starvation- and 5-FU-induced apoptosis. HEK293 cells were transfected with empty vector (no), REP1 wild-type (WT) or REP1 mutant (Mut). (a) Expressions of FLAG (REP1), BIM, BAK, and p27 were analyzed by immunoblotting. β-ACTIN was included as an internal loading control. Numbers below blots indicate the expression as measured by fold change. (b) Growth rate of transfected cells in serum-starvation culture (0.1% FBS). Cells were harvested at the indicated times and counted after trypan blue staining to exclude dead cells. (c) The frequency of apoptotic (active caspase-3+) cells among the transfected cells cultured in serum-starvation condition (0.1% FBS) was estimated by flow cytometry analysis. (d) Transfected cells were treated with 50 uM of 5-FU for 24 h and then the frequency of apoptotic (active caspase-3+) cells was determined by flow cytometry analysis. All graphs represent two independent experiments performed in triplicate. Error bars represent S.D. from the mean. *P<0.01, **P<0.001