Figure 2: BCLAF1-L is required for survival of colon cancer cells.

(a) RKO cells were treated for 72 h with the indicated siRNAs. BCLAF1-L knockdown was assessed by western blotting using β-actin as a loading control. (b) Cell proliferation assay. RKO cells were infected with retroviruses expressing shRNA-BCLAF1-L1, L2 or control shRNA, and selected for puromycin resistance. Cell proliferation was measured by trypan blue exclusion. (c,d) Clonogenic survival assay and quantification of RKO and HCT116 cells either stably expressing shLuci or each of shRNA-L1 and L2 as described in b. Three independent experiments were performed and representative cells stained with crystal violet were shown. The number of focal adhesions was quantified in the right bar graph and results are shown as mean±s.d. (e) RKO cells were transfected with GFP-BCLAF1-L plasmid and selected for neomyocin resistance. Overexpression of BCLAF1-L was confirmed by western blotting. (f,g) Clonogenic survival assay and quantification was performed with RKO cells stably expressing GFP-BCLAF1-L as described in c,d. All experiments were repeated three times and results are shown as mean±s.d.