Journal: PLoS ONE

Article Title: miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

doi: 10.1371/journal.pone.0070192

Figure Lengend Snippet: (A) Effects of miR-29c on endogenous integrin β1 protein. Changes in protein expression of Int β1 was determined by Western blotting analysis in 95D 29c , 95D MiNC, 95C 29ci and 95C IhNC. GAPDH was used as an internal loading control. (B) The gray levels of Western blotting are shown by bar gragh. miR-29c mimics inhibit endogenous integrin β1 in 95D cell lines (95D 29c and 95D MiNC), while miR-29c inhibitors promote integrin β1 protein expression. ** p <0.01 (Student's t -test, n = 3). (C) Effects of miR-29c on endogenous MMP2 protein levels by Western blotting. (D) The gray levels of Western blotting are shown by bar gragh. ** p <0.01 (Student's t -test, n = 3). (E) Effects of miR-29c on MMP2 and MMP9 enzyme activity in lung cancer by gelatin zymography. (F) The gray level of every band was measured to check the differences in MMP2 enzyme activity in 95C and 95D cells (The brightest band [95D MiNC] was set to 1 unit to compare with other gray levels). ** p <0.01 (Student's t -test, n = 3). (G) The gray level of every band was measured to check the difference in MMP9 enzyme activity between 95C and 95D cells (The brightest band [95D MiNC] was set to 1 unit to compare with other gray levels). ** p <0.01 (Student's t -test, n = 3).

Article Snippet: The membranes were blocked in 5% non-fat milk in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated overnight at 4°C by the appropriately diluted primary antibodies for rabbit monoclonal anti-human integrin β1 and MMP2 (diluted 1∶1000; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Control, Activity Assay, Zymography

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Rabbit anti-Akt (#9272S), anti-phospho-Akt (Ser473) (#9271L), anti-Src (#2110S), anti-phospho-Src (#6943S), anti-FAK (#3285S), anti-phospho-FAK (Y397) (#3281S), anti-integrin β1 (#9699S), anti-integrin β2 (#73663S), anti-integrin β3 (#13166S), anti-integrin α5 (98204S), anti-Sp1 (#9389S) and mouse anti-β-actin (#3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Rabbit anti-Akt (#9272S), anti-phospho-Akt (Ser473) (#9271L), anti-Src (#2110S), anti-phospho-Src (#6943S), anti-FAK (#3285S), anti-phospho-FAK (Y397) (#3281S), anti-integrin β1 (#9699S), anti-integrin β2 (#73663S), anti-integrin β3 (#13166S), anti-integrin α5 (98204S), anti-Sp1 (#9389S) and mouse anti-β-actin (#3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Rabbit anti-Akt (#9272S), anti-phospho-Akt (Ser473) (#9271L), anti-Src (#2110S), anti-phospho-Src (#6943S), anti-FAK (#3285S), anti-phospho-FAK (Y397) (#3281S), anti-integrin β1 (#9699S), anti-integrin β2 (#73663S), anti-integrin β3 (#13166S), anti-integrin α5 (98204S), anti-Sp1 (#9389S) and mouse anti-β-actin (#3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR