Journal: PLoS ONE

Article Title: Silencing of the hTERT Gene by shRNA Inhibits Colon Cancer SW480 Cell Growth In Vitro and In Vivo

doi: 10.1371/journal.pone.0107019

Figure Lengend Snippet: ( A ) Endogenous hTERT mRNA expression in SW480, DLD-1 and HT29 cell lines. There were significant differences among SW480, HT29 and DLD-1 cells ( # P <0.01), and between SW480 and HT29 cells (* P <0.05). ( B ) Potency of hTERT siRNAs in SW480 cells. Levels of hTERT mRNA were significantly lower in the cells transfected with hTERT siRNAs than in cells transfected with negative control (NC) shRNA or Lipofectamine alone (Lipo). * P <0.05, # P <0.01, compared with the NC-shRNA and Lipo groups. Results are mean ± SD from three independent experiments.

Article Snippet: Transfections were performed using Lipofectamine reagent (GenePharma Co., Shanghai, China) according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Negative Control, shRNA

Journal: PLoS ONE

Article Title: Silencing of the hTERT Gene by shRNA Inhibits Colon Cancer SW480 Cell Growth In Vitro and In Vivo

doi: 10.1371/journal.pone.0107019

Figure Lengend Snippet: ( A ) RT-PCR analysis of hTERT mRNA expression in SW480 cultures after mock-transfection with culture medium alone (Blank), Lipofectamine alone (Lipo), negative control siRNA (NC-shRNA), or hTERT-shRNA. Levels of mRNA levels were significantly lower after 48-h transfection with hTERT-shRNA than after control transfections. ▴ P <0.05, # P <0.01, compared with the Blank; * P <0.05, compared with Lipo and NC-shRNA. ( B ) Immunocytochemistry of hTERT protein expression. (a) Blank cells, (b) Lipo cells, (c) NC-shRNA cells, and (d) hTERT-shRNA cells. All views, ×400. Higher greyscale value indicates lower protein level. Protein levels were significantly lower after 48-h transfection with hTERT-shRNA than after control transfections. # P <0.01, compared with the other groups; * P <0.05, compared with the Blank. ( C ) HTERT down-regulation inhibits telomerase activity. Telomerase activity [adjusted absorbance (450 nm–630 nm)] was significantly lower in cells transfected with hTERT-shRNA than in control cells. * P <0.05, compared with the Lipo and Blank controls. Results are mean ± SD from three independent experiments.

Article Snippet: Transfections were performed using Lipofectamine reagent (GenePharma Co., Shanghai, China) according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control, shRNA, Control, Immunocytochemistry, Activity Assay

Journal: PLoS ONE

Article Title: Silencing of the hTERT Gene by shRNA Inhibits Colon Cancer SW480 Cell Growth In Vitro and In Vivo

doi: 10.1371/journal.pone.0107019

Figure Lengend Snippet: ( A ) hTERT-shRNA reduces growth and proliferation of SW480 cells. The proportion of cells in G0/G1 phase was significantly higher and the proliferation index was much lower after transfection with hTERT-shRNA than after control transfections. * P <0.05, compared with the other three groups; # P <0.05, compared with NC-shRNA. ( B ) hTERT-shRNA increases apoptosis of SW480 cells. Cells were transfected with (a) hTERT-shRNA or (b) NC-shRNA, stained with diaminobenzidine (brown) and counter-stained with hematoxylin (blue) to label nuclei. Images are representative results from three independent experiments. Magnification, ×400. The apoptotic index (AI) was significantly higher in cells transfected with hTERT-shRNA. # P <0.01, compared with the other three groups. ( C ) Transmission electron microscopy analysis of SW480 cells transfected with (a-c) hTERT-shRNA or (d) NC-shRNA. Apoptotic morphology was visible in cells transfected with hTERT-shRNA. Magnification, ×3800. ( D ) Rhodamine 123 fluorescence intensity analysis of SW480 cells transfected with (a) culture medium alone (Blank), (b) Lipofectamine alone (Lipo), (c) NC-shRNA or (d) hTERT-shRNA. Stained cells were analyzed by confocal scanning microscopy; higher fluorescence intensity indicates lower mitochondrial membrane potential. Magnification, ×400. Cells transfected with hTERT-shRNA showed significantly higher rhodamine 123 fluorescence than did control cells. * P <0.05, compared with NC-shRNA or Lipo; # P <0.01, compared with Blank. Results are the mean ± SD from three independent experiments.

Article Snippet: Transfections were performed using Lipofectamine reagent (GenePharma Co., Shanghai, China) according to the manufacturer’s instructions.

Techniques: shRNA, Transfection, Control, Staining, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Membrane