Journal: Carcinogenesis

Article Title: Differential role of Sloan-Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-?)-induced Smad signaling in prostate cancer cells

doi: 10.1093/carcin/bgs252

Figure Lengend Snippet: Effects of Nodal and TGF-β on cell proliferation and migration of prostate cell lines. (A and B). The effects of Nodal and TGF-β on DNA synthesis in PZ-HPV7, LNCaP, DU145 and PC3 cells as determined by 3H-thymidine incorporation assay. The cells were serum-starved for 24h and treated with different concentrations of rhNodal and TGF-β for 18h in the presence of 5% FBS. The cells were then pulse labeled for 4h with 1 µCi/ml 3H-thymidine and DNA incorporated radioactivity was determined by liquid scintillation counting. Each bar represents mean ± SEM (n = 3). *Significantly different compared with untreated controls. (C and D) Nodal and TGF-β dose-dependently induced migration in PC3 cells but not in DU145 cells in a transwell migration assay. Representative images of DU145 and PC3 cell lines after different treatments. Cells were visualized under 10× objectives. EGF (3ng/ml) was used as a positive control. Each bar represents mean ± SEM (n = 3). *Significantly different (P < 0.05) compared with untreated controls.

Article Snippet: Prostate epithelial stem cell line (WPE), immortalized normal PrECs (PZ-HPV7), immortalized prostate luminal epithelial cell line (RWPE1), k-ras transformed RWPE1 cell line (RWPE2) and prostate cancer cell lines (LNCaP, DU145 and PC3) were obtained from American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Migration, DNA Synthesis, Thymidine Incorporation Assay, Labeling, Radioactivity, Transwell Migration Assay, Positive Control

Journal: Carcinogenesis

Article Title: Differential role of Sloan-Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-?)-induced Smad signaling in prostate cancer cells

doi: 10.1093/carcin/bgs252

Figure Lengend Snippet: Activation of Nodal and TGF-β signaling in prostate cell lines. (A) Western blot analyses of phosphorylated Smad2 and Smad3 and β-actin in PZ-HPV7, DU145 and PC3 cells at different time periods after treatment with Nodal (200ng/ml) or TGF-β1 (5ng/ml). Western blots using anti-β-actin antibody were used as internal controls. Quantitative analysis of p-Smad2 and p-Smad3 in PZ-HPV7, DU145 and PC3 cells treated with Nodal and TGF-β were relative to that of the untreated control (designated as one) after normalization to the signal obtained with Smad2/3. Each bar represents mean ± SEM (n = 3). *Significantly different (P < 0.05) compared with untreated controls. (B) Western blot analyses of phosphorylated Smad2 and Smad3, total Smad2/3 and β-actin in PC3 cells after pretreatment with Smad3 inhibitor (SIS3, 1 µM). (C) Pretreatment with SIS3 (1 µM) for 30min completely blocked the migration of PC3 cells induced by TGF-β (5ng/ml) but not in Nodal (500ng/ml) and EGF (3ng/ml) treated cells. The data were presented as mean ± SEM (n = 3). *Significantly different (P < 0.05) compared with untreated controls.

Article Snippet: Prostate epithelial stem cell line (WPE), immortalized normal PrECs (PZ-HPV7), immortalized prostate luminal epithelial cell line (RWPE1), k-ras transformed RWPE1 cell line (RWPE2) and prostate cancer cell lines (LNCaP, DU145 and PC3) were obtained from American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Activation Assay, Western Blot, Control, Migration

Journal: Carcinogenesis

Article Title: Differential role of Sloan-Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-?)-induced Smad signaling in prostate cancer cells

doi: 10.1093/carcin/bgs252

Figure Lengend Snippet: Basal expression of Ski in prostate cell lines. (A) Total RNAs were isolated and semi-quantitative RT–PCR was performed to determine the mRNA levels of Ski in prostate cell lines. L-19 was used as an internal control. No RT samples derived from the same RNAs were also included. (B) Western blot analysis of Ski protein levels in prostate cell lines. Total cellular proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted using anti-Ski antibody. (C) Ski (green) expression in PZ-HVP7, DU145 and PC3 prostate carcinoma cells. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Ski was predominately localized in cytoplasm of the cells; the basal expression of Ski was high in prostate cancer cell lines but low in PZ-HPV7 cells. (D) Western blot analyses of Ski in PZ-HVP7 and PC3 cells treated with MG132 (25 µM). Anti-β-actin antibody was served as internal controls. Quantitative analysis of Ski in PZ-HPV7 and PC3 cells with or without treatment with MG132 (25 µM) (lower panel) were relative to untreated control (designated as one) after normalization to the signal obtained with β-actin. Each bar represents mean ± SEM (n = 3). *Significantly different (P < 0.05) compared with untreated controls. (E) Tissue sections were deparaffinized, rehydrated and blocked for endogenous peroxidase. Sections were blocked using normal goat serum in PBS and incubated with Ski antibody overnight. Sections were washed and incubated with Alexa Flour 488 anti-rabbit for 30min, washed again, and incubated with 4′,6-diamidino-2-phenylindole. Sections were then washed and counterstained with hematoxylin and mounted using xylene mounting medium. Immunofluorescence and H&E staining were analyzed by Zeiss microscope using 40× magnification. Representative images of the control and cancer tissues are shown.

Article Snippet: Prostate epithelial stem cell line (WPE), immortalized normal PrECs (PZ-HPV7), immortalized prostate luminal epithelial cell line (RWPE1), k-ras transformed RWPE1 cell line (RWPE2) and prostate cancer cell lines (LNCaP, DU145 and PC3) were obtained from American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Derivative Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Incubation, Immunofluorescence, Staining, Microscopy