Figure 3.

miR-133b perturbs mitotic timing by downregulating Nup214. (a) and (b) miR-133b halts cells at G2/M phase. UPCI:SCC084 cells were transiently transfected with 1 μg pSB-miR-133b or empty vector, synchronized and harvested at 0, 4, 6, 8 and 10 h from second thymidine release. DNA content was analysed by flow cytometry. (a) shows representative images while (b) shows the percentage of cells in each phase at each time-point. (c) and (g) Representative pictures showing miR-133b delays cyclinB1, cyclinA degradation and H3 dephosphorylation while Nup214 rescues the normal pattern even in presence of miR-133b. UPCI:SCC084 (c) and HCT116 (g) 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. Cell lysates were prepared at 0, 4, 6, 8, 10 and 12 h from second thymidine release followed by Western blot with antibodies against cyclinB1, cyclinA, p-H3, Nup214 and β-actin. (d), (e), (f), (h), (i) and (j) Densitometric values of bands by ImageJ are plotted: cyclinB1 (d, h) and cyclinA (f, j) were normalized to β-actin. P-H3 (e, i) was normalized to total H3. All three proteins were further normalised to the respective protein expressions at 0 h in empty vector-treated cells. For (a), (c) and (g), images are representative of three different experiments while for (d), (e), (f), (h), (i) and (j), data represent three independent experiments and shown as average ± S.D. Black arrow in (g) represents a non-specific band.