Figure 2
From: REP1 inhibits FOXO3-mediated apoptosis to promote cancer cell survival
Silencing of REP1 sensitizes colon cancer cells to serum starvation- and 5-FU-induced apoptosis. (a) Immunohistochemical staining of REP1 was performed on tissue microarray of human colon cancer specimens. Representative images of immunohistochemical staining of REP1 in colon tissues from normal and carcinoma patients. High-magnification images are shown in inset. Scale bar, 100 μm. (b) Protein expression of REP1 in normal colon cells (FHC) and colon cancer cells (LoVo, SNUC4, HT29, HCT116) was determined by immunoblotting. β-ACTIN was included as an internal loading control. Numbers below blots indicate the expression as measured by fold change. (c–e) HCT116 and LoVo cells were transfected with siRNAs targeting GFP or REP1. (c) Growth rate of siGFP- versus siREP1-transfected HCT116 and LoVo 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. (d) Flow cytometry analysis of the frequency of apoptotic (active caspase-3+) cells among siGFP- or siREP1-transfected HCT116 and LoVo cells. Isotype control staining is indicated by solid gray regions and anti-active caspase-3 staining is indicated by black dotted-lines. (e) Expressions of REP1, BIM, BAK, BID, BAX, BAD, 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. (f and g) HCT116 and LoVo cells were transfected with siRNAs targeting GFP or REP1. Cells were treated with the indicated concentrations of 5-FU for 24 h. (f) Viability of siGFP- or siREP1-transfected HCT116 and LoVo was measured by an MTT assay, and then the concentrations resulting in 50% inhibition of cell viability (IC50 values) were determined. Each experiment was performed in triplicate, and error bars represent S.D. from the mean. (g) Flow cytometry analysis of the frequency of apoptotic (active caspase-3+) cells among siGFP- or siREP1-transfected HCT116 and LoVo cells. Isotype control staining is indicated by solid gray regions and anti-active caspase-3 staining is indicated by black dotted-lines. All Graphs represent two independent experiments performed in triplicate. Error bars represent S.D. from the mean. *P<0.01, **P<0.001
