Figure 3
BIM is induced by miR-491-5p and is a critical determinant in miRNA-mediated apoptosis in IGROV1-R10 cells. All data presented were obtained 72 h after transfection of IGROV1-R10 or SKOV3 cells with miR-491-5p or miRNA-Ctrl (miR-Ctrl). (a) Protein expression levels of the BH3-only BIM and PUMA and the anti-apoptotic BCL-XL were assessed by western blotting. Changes in migration on SDS-PAGE of BIM were indicated with an arrowhead. (b). Lysates from miR-Ctrl or miR-491-5p transfected IGROV1-R10 cells were treated or not with phosphatase alkaline and analysed by western blotting for changes in migration of BIM in SDS-PAGE. Changes in phosphorylation on SDS-PAGE of BIM were indicated with an arrowhead. (c) IGROV1-R10 cells were harvested, lysed and subjected to immunoprecipitation (IP) with BIM or MCL1 antibodies. The resulting immune complexes as well as total lysates were analysed by western blotting with BIM or MCL1 antibodies. The relative densitometry values (calculated relative to control conditions after normalization with β-actin) were determined using ImageJ software and are shown on the bottom of each western blot. (d and e) IGROV1-R10 cells were transfected concomitantly with siRNA against BIM (siRNA-BIM) or siRNA control and indicated miRNAs. (d) Seventy-two hours after transfection, cells were subjected to flow cytometry to determine the percentage of sub-G1 population (data are presented as mean±S.D. of triplicate experiments). A representative DNA content histogram obtained by flow cytometry is also shown. (e) Cells were lysed and subjected to western blot analysis to determine BIM, BCL-XL, caspases-3 (pro and cleaved forms) protein expression levels. β-actin level is shown as loading control
