Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 1. Expression of signal transducers and activators of transcription 3 (STAT3) in SW1990 cells and HeLa cells. (a) STAT3 expression was determined by Western blot analysis. Detection of β-actin was used as a loading control. HeLa cell line served as a positive control of STAT3 expression. (b) STAT3 distribution in SW1990 cells was examined by immunocytochemistry (400 ×), the buffy particle corresponded for STAT3 (shown by arrow).

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Positive Control, Immunocytochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 2. Effects of signal transducers and activators of transcription 3 (STAT3) specific shRNA expression vector on STAT3 expression. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3- siRNA-II and pRNAT-STAT3-siRNA-III were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT- PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3-siRNA-II and pRNAT-STAT3-siRNA-III. The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was deter- mined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. Results shown are for one represent- ative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: shRNA, Expressing, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Membrane, Control

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 3. Effects of stable transfection of pRNAT-STAT3-siRNA-II vector on signal transducers and activators of transcription 3 (STAT3) expres- sion. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was determined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. (c) STAT3 DNA-binding activity. Cell nuclear protein extracts (10 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) and subjected to electrophoretic mobility shift analysis (EMSA) using biotin end-labeled oligonucleotide probes containing a consensus-binding motif for STAT3. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Stable Transfection, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot, Expressing, Membrane, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 4. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell invasion in vitro. SW1990-RNAi-a, SW1990-RNAi-b, SW1990-Con and the parental cell lines were seeded into the upper compart- ments of invasion chambers. Cells were allowed to invade for 48 h at 37°C. The tumor cells that invaded the ECMatrix and migrated through the polycarbonate membrane were stained by the staining solution and dissolved in 10% acetic acid. The dye/solution mixture was transferred onto a 96-well plate for colorimetric reading of optical density (OD) at 560 nm. The number of migrated cells that penetrated through the ECMatrix-coated filters was expressed as the OD at 560 nm. The standard deviation bars represent replicates within the assay. *P < 0.001 compared with that of parental SW1990 cells. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vitro, Membrane, Staining, Standard Deviation

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 5. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell metastasis in vivo. In total, 5 × 105 SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were injected i.v. into groups of nude mice (n = 6). The mice were killed on day 21 or when mice became moribund. The number of experimental lung metastases was determined with the aid of a dissecting microscope. Results were expressed as the median number and range of lung metastasis nodules. *P < 0.001 compared with that of parental SW1990 cells. Results shown here are for one representative experiment of three. (a) H&E staining of formalin-fixed, paraffin-embedded lung tissue of nude mice (100 ×). The metastasis nodus are indicated by arrows. (b) H&E staining of formalin-fixed, paraffin-embedded metastasis nodus of lung tissue of nude mice (400 ×). (c) In SW1990 cells injected nude mice, STAT3, matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF) distribution in metastasis nodus of lung tissue was examined by immunohistochemistry (400 ×), the buffy particle corresponded for STAT3, MMP-2 and VEGF. Immunohistochemistry showed that STAT3, MMP-2 and VEGF were distributed in cytoplasm.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vivo, Transfection, Control, Plasmid Preparation, Injection, Microscopy, Staining, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 6. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on the expression of matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF). (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3- RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for MMP-2, VEGF, and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of MMP-2 protein was determined using Western blot analysis with an anti-MMP-2 antibody. The expression of VEGF protein was determined by hybridizing the same membrane filter with an anti-VEGF antibody. The levels of β-actin expression were determined as a control for the equivalent protein loading. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Western Blot, Membrane