Figure 4

Smad7 knockdown in CRC cells enhances eIF2α phosphorylation and downregulates CDC25A expression. (a) Transfection of HCT-116 cells with Smad7 antisense oligonucleotide (AS) reduces CDC25A protein expression. HCT-116 cells were either left untreated (Untr) or transfected with Smad7 AS or Smad7 sense oligonucleotide (S). After 24 h cells were washed with PBS and cultured for further 24 h. CDC25A, CDC25B and CDC25C expression was assessed by Western blotting. One of three representative experiments in which similar results were obtained is shown. (b) Smad7 AS treatment does not reduce CDC25A RNA expression. HCT-116 cells were either left untreated (Untr) or transfected with Smad7 AS or S. CDC25A transcripts were evaluated by real-time polymerase chain reaction. Levels are normalized to β-actin. Values are mean±S.D. of three experiments. Smad7 AS-transfected cells versus Smad7 S-transfected cells, *P<0.001. (c) CDC25A protein downregulation in cells treated with Smad7 AS is not reverted by proteasome inhibitors (MG115 and MG132). Representative Western blots for CDC25A and β-actin in extracts of HCT-116 cells transfected with either Smad7 S or AS and then cultured in the presence or absence of MG115 and MG132 for 24 h. One of three representative experiments in which similar results were obtained is shown. (d) Treatment of CRC cells with Smad7 AS enhances eIF2α phosphorylation and this phenomenon precedes the downregulation of CDC25A expression. Representative Western blots for p-eIF2α Ser51, eIF2α, CDC25A and β-actin in extracts of HCT-116 cells transfected with either S or AS and then cultured for the indicated time points. One of 3 three representative experiments in which similar results were obtained is shown. (e) Treatment of CRC cells with Smad7 AS reduces de novo protein synthesis. Representative western blots for puromycin and β-actin in extracts of HCT-116 cells transfected with either Smad7 S or AS and then cultured for 24 h. Where indicated, cells were pulsed with puromycin. One of three representative experiments in which similar results were obtained is shown