Figure 6
miR-133b-mediated mitotic delay leads to cell death. (a) and (b) Ectopic miR-133b affects clonogenicity of cells. UPCI:SCC084 cells were seeded at a density of 103 and transiently transfected with 0, 0.25, 0.5, 1 and 2 μg pSB-miR-133b. Colonies were stained with methylene blue after a week. (a) Representative images are shown. (b) Colonies were counted from (a) and plotted as shown. (c) and (d) Excess miR-133b induces apoptosis. 105 UPCI:SCC084 cells were transiently transfected with empty vector (1 μg), pSB-miR-133b (1 μg), or pSB-miR-133b (1 μg) and pCMV-Myc-CAN/Nup214 (1 μg). At 0, 24, 48 and 72 h post-transfection, cells were subjected to annexin V-FITC/PI staining followed by flow cytometry analysis. (e) Ectopic miR-133b elevates cleaved caspase3 levels. 105 UPCI:SCC084 cells were transiently transfected with empty vector (1 μg), pSB-miR-133b (1 μg), or pSB-miR-133b (1 μg) and pCMV-Myc-CAN/Nup214 (1 μg). Cells were fixed, permeabilized and stained with control antibodies or cleaved caspase3 antibody at 0 and 72 h post-transfection followed by flow cytometric analysis. Colour codes of histograms and mean FITC values are indicated. For (b)-(d), data represent three independent experiments and shown as average ± S.D. For (a) and (e), images are representative of three independent experiments.