Figure 4

miR-133b modulates mitotic timing. (a) and (b) HCT116 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) and synchronized. At 4, 6, 8 and 10 h from second thymidine release, cells were fixed, stained with DAPI and visualized under a fluorescence microscope. Representative images are shown (a). Only brightly-stained (FITC) cells were considered for MI evaluation. Scale bar represents 10 μm. Images represent 40× magnification. MI were calculated and plotted as shown (b); represents three independent experiments and shown as average ± S.D. (c) HeLa-H4-pEGFP stable 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) and synchronized. Medium was replaced with phenol red-free medium and visualized under time lapse fluorescence microscope. ‘0.00’ time-point is that time (8 h post-release) when chromosomes for each of the three treated condition were found to be aligned at the metaphase plate. Representative frozen images are shown and time stamps indicated. Scale bar represents 5 μm. Images represent 63× magnification.