Figure 7
NUP214 knockdown by siRNAs attests miR-133b-induced phenotypes. (a) Representative picture showing Nup214 knockdown by specific siRNAs. UPCI:SCC084 cells were transiently transfected with scrambled siRNA (80 nM), Nup214-specific siRNAs (80 nM), or Nup214-specific siRNAs (80 nM) and pCMV-Myc-CAN/Nup214 (1 μg). Cell lysates were prepared followed by Western blot with antibodies against Nup214 and β-actin. (b) Densitometric values of bands were normalized to those of β-actin by ImageJ and plotted. For (a) and (b), data represent three independent experiments and shown as average ± S.D. (c) Nup214 knockdown represses colony forming potential of cells. UPCI:SCC084 cells were seeded at a density of 103 and transiently transfected with scrambled siRNA (80 nM), Nup214-specific siRNAs (80 nM), or Nup214-specific siRNAs (80 nM) and pCMV-Myc-CAN/Nup214 (1 μg). Colonies were stained with methylene blue after a week, counted and plotted as shown. (d) and (e) Nup214 knockdown induces apoptosis. 105 UPCI:SCC084 cells were transiently transfected with scrambled siRNA (80 nM), Nup214-specific siRNAs (80 nM), or Nup214-specific siRNAs (80 nM) and pCMV-Myc-CAN/Nup214 (1 μg). At 0 and 72 h post-transfection, cells were subjected to annexin V-FITC/PI staining followed by flow cytometry analysis. Representative images are shown (d) and data plotted (e). (f) and (g) Nup214 knockdown elevates cleaved PARP levels. 105 UPCI:SCC084 cells were transiently transfected with scrambled siRNA (80 nM), Nup214-specific siRNAs (80 nM), or Nup214-specific siRNAs (80 nM) and pCMV-Myc-CAN/Nup214 (1 μg). Cells were fixed, permeabilized and stained with control antibodies or cleaved PARP antibody at 0 and 72 h post-transfection followed by flow cytometric analysis. Color codes of histograms are indicated in representative images (f). Data was plotted as shown (g).