Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: Relationships between the expression of miR-126, CXCR4, and RhoA signaling pathway components and colon cancer clinicopathological features

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: ( A ) The relative levels of CXCR4 and the RhoA signaling pathway components RhoGEF, ARHGAP5, PI3K, ROCK, PAK, and PKN were assessed in colon cancer tissues and in paired normal colonic mucosa by immunohistochemical staining. Representative images are shown (200×). ( B ) Kaplan-Meier survival curves are shown for patients according to the expression of CXCR4, RhoA, RhoGEF, and ROCK in their colon cancer tissues. Colon cancer-associated death was significantly correlated with expression of CXCR4, RhoA, RhoGEF, and ROCK. Decreased patient overall survival was associated with higher expression of CXCR4 (upper left panel; n = 50/66; p = 0.023), RhoA (upper right panel; n = 47/69; p = 0.0192), RhoGEF (lower left panel; n = 56/67; p = 0.022), and ROCK (lower right panel; n = 48/69; p = 0.001). The red lines indicate patients with cancer tissues expressing CXCR4, RhoA, RhoGEF, and ROCK; blue lines indicate the patient tissues not expressing these proteins. In each graph, each ‘ N ’ implies negative expression, and each ‘ P ’ implies positive expression.

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: Correlation among the expression of miR-126, CXCR4, and components of the RhoA signaling pathway

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: ( A ) The mRNA levels of CXCR4 and components of the RhoA signaling pathway were determined by qRT-PCR in transfected HCT116 and SW480 cells. GAPDH expression served as the internal control.* p < 0.05; ** p < 0.01; *** p < 0.001. ( B ) Western Blot for the detection of CXCR4 and components of the RhoA signaling pathway in transfected HCT116 and SW480 cells. GAPDH served as the internal control. ( C ) RhoA G-LISA activation assays were used to measure RhoA activity in the transfected cells. *** p < 0.001.

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Control, Western Blot, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: ( A ) Western Blots were used to confirm CXCR4 expression. Transient transfection of HCT116/miR-126 cells with CXCR4 plasmids resulted in increased expression of CXCR4 compared with that cells transfected with unmodified vectors or not transfected (upper panels). AMD3100 was used to inhibit CXCR4 expression in SW480/anti-miR-126 cells (bottom panels). At a concentration of 1000 ng/ml AMD3100, CXCR4 expression was substantially reduced. ( B , C ) Wound-healing assays were used to measure cell migration capacity. Migration increased following transient transfection with the CXCR4 plasmids (left panels), but the migration of SW480/anti-miR-126 cells was inhibited after exposure to AMD3100 (right panels) * p < 0.05, *** p < 0.001. ( D ) Transwell assays were used to measure cell invasion capacity. The invasion capacity of HCT116/miR-126 cells increased following transient transfection with the CXCR4 plasmids (top). The red, blue, and purple bars denote CXCR4 + HCT116/miR-126, vector + HCT116/miR-126, and vector + HCT116/miR-NC, respectively. The invasion capacity of SW480/anti-miR-126 cells was reduced after exposure to AMD3100 (bottom). The red, blue, and purple bars denote AMD3100 + SW480/anti-miR-126, PBS + SW480/anti-miR-126, and PBS + SW480/anti-miR-NC, respectively. Panels on the left show stained cells that had invaded. Cells in five randomly selected areas were counted, and a statistical analysis was performed using SPSS 17.0. ( E ) MTT assays were used to measure cell proliferation. The proliferation of HCT116/miR-126 cells was increased after transient transfection with the CXCR4 plasmids (top panel). The proliferation of SW480/anti-miR-126 cells was reduced after exposure to AMD3100 (bottom panel). The data represent the mean ± SD of three replicates ** p < 0.01, *** p < 0.001.

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Transfection, Concentration Assay, Migration, Plasmid Preparation, Staining

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: ( A ) Western blot analysis of the expression levels of components of the RhoA signaling pathway. Left panels show expression levels in HCT116/miR-126 cells after transient transfection with the CXCR4 plasmids. Right panels show expression levels in SW480/anti-miR-126 cells after exposure to AMD3100 (1000 ng/ml). ( B ) RhoA activation G-LISA assays were used to measure RhoA activity in HCT116/miR-126 cells transiently transfected with the CXCR4 plasmids (left panels) and in SW480/anti-miR-126 cells exposed to 1000 ng/ml AMD3100 (right panels). * p < 0.05, *** p < 0.001.

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Transfection, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: ( A ) Western Blot showing the levels of Gα 12 and Gα 13 in transfected HCT116 and SW480 cells. GAPDH served as the internal control. ( B , C ) In vitro co-immunoprecipitation (co-IP) confirmed the interactions between CXCR4, RhoA, and the Gα 12 /Gα 13 complex and revealed a complex composed of CXCR4 and Gα 13 . Total protein was extracted from untransfected HCT116 and SW480 cells. Total (input) and eluted (co-IP) proteins were subjected to Western Blot, which was performed with anti-Gα 12 , anti-RhoA, and anti-Gα 13 . ( D ) Western Blot was used to confirm the expression of Gα 13 (right), and the G-LISA assay was used to measure RhoA activity in HCT116 cells after knocking down Gα 13 expression with siRNA-Gα 13 (left). The control group was exposed to the non-specific siRNA, ** p < 0.01.

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection, Control, In Vitro, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Activity Assay

Journal: Oncotarget

Article Title: MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

doi: 10.18632/oncotarget.11176

Figure Lengend Snippet: Rho GDP-dissociation inhibitors (RhoGDIs) are important regulators of the Rho family of small GTPases. RhoGDIs can prevent RhoA activation by inhibiting GDP dissociation from RhoA. RhoGEFs activate RhoA, and ARHGAP5 can negatively regulate RhoA. PAK, PKN, ROCK, and PI3K are downstream effectors of RhoA signaling. MiR-126 inhibits cell proliferation and migration via its regulation of the CXCR4/Gα 13 /RhoA signaling axis in colon cancer pathology.

Article Snippet: Rabbit polyclonal primary antibodies against CXCR4 (sc-9046), Gα 12 (sc-409), and Gα 13 (sc-410), mouse monoclonal antibodies against GAPDH (sc-166545), RhoA (sc-418), PAK (sc-166174), PKN (sc-136037), PI3K (sc-166365), ROCK (sc-17794), and RhoGEF (sc-166301), and the horseradish peroxidase–conjugated secondary antibodies against rabbit-IgG (sc-2004) and mouse-IgG (sc-2005) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Migration

Journal: Journal of Translational Medicine

Article Title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

doi: 10.1186/1479-5876-10-68

Figure Lengend Snippet: CXCR4 and CXCR7 expression in human formalin-fixed paraffin-embedded pancreatic cancer specimens . Pancreatic cancer tissue was obtained from patients who had undergone resection for pancreatic adenocarcinoma with Institutional Review Board approval. Tissue blocks were sectioned (5 μm) and deparaffinized with xylene. Antigen retrieval was performed and one section was incubated with the anti-CXCR4 antibody and the next consecutive section from the same tissue block was incubated with the anti-CXCR7 antibody.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against phospho-ERK1/2 and total ERK1/2 (Cell Signaling; Beverly, MA), rabbit polyclonal antibodies against CXCR4 (Abcam, Cambridge, MA) and CXCR7 (Abcam and R&D Systems), a goat polyclonal antibody against β-arrestin-2 (Abcam), and a mouse monoclonal antibody against K-Ras (Calbiochem; San Diego, CA).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Incubation, Blocking Assay

Journal: Journal of Translational Medicine

Article Title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

doi: 10.1186/1479-5876-10-68

Figure Lengend Snippet: CXCR4 and CXCR7 expression in pancreatic cancer cell lines . Immunoblotting was performed for CXCR4 and CXCR7 protein expression in established human pancreatic cancer cell lines. HT29 colon cancer cells were used as a negative control for CXCR7 expression. GAPDH was used as a loading control.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against phospho-ERK1/2 and total ERK1/2 (Cell Signaling; Beverly, MA), rabbit polyclonal antibodies against CXCR4 (Abcam, Cambridge, MA) and CXCR7 (Abcam and R&D Systems), a goat polyclonal antibody against β-arrestin-2 (Abcam), and a mouse monoclonal antibody against K-Ras (Calbiochem; San Diego, CA).

Techniques: Expressing, Western Blot, Negative Control, Control

Journal: Journal of Translational Medicine

Article Title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

doi: 10.1186/1479-5876-10-68

Figure Lengend Snippet: The effect of CXCL12 exposure on MAPK signaling . a) Pancreatic cancer cells PANC-1 and MiaPaCa2 were serum-starved and then exposed to CXCL12 (100 ng/ml) and CXCL11 (200 ng/ml) over a range of time periods (5 min to 1 h). Immunoblotting was performed for phosphorylated ERK1/2. Total ERK1/2 and GAPDH were used as loading controls. b) PANC-1 cells were transfected with control, CXCR4, or CXCR7 siRNA (100 nM) using RNAiMAX (10 μl). The immunoblots verify down-regulation of CXCR4 and CXCR7 expression. c) Following transfection, the cells were serum-starved and then exposed to CXCL12 (100 ng/ml). Immunoblotting was performed for phospho-ERK1/2. Total ERK1/2 and GAPDH were used as loading controls.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against phospho-ERK1/2 and total ERK1/2 (Cell Signaling; Beverly, MA), rabbit polyclonal antibodies against CXCR4 (Abcam, Cambridge, MA) and CXCR7 (Abcam and R&D Systems), a goat polyclonal antibody against β-arrestin-2 (Abcam), and a mouse monoclonal antibody against K-Ras (Calbiochem; San Diego, CA).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: Journal of Translational Medicine

Article Title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

doi: 10.1186/1479-5876-10-68

Figure Lengend Snippet: K-Ras activity in response to CXCL12 exposure . a) Pancreatic cancer cells were serum-starved and then exposed to CXCL12 (100 ng/ml) and CXCL11 (200 ng/ml) for 15 min. Whole cell lysates were assessed for K-Ras activity using a Raf pull-down assay. b) Cells were transfected with control, CXCR4, or CXCR7 siRNA (100 nM). Following serum-starvation cells were treated with CXCL12 (100 ng/ml) for 15 min. K-Ras activity was normalized to each untreated baseline level; and relative increases are depicted. The mean absorbance ± one SD was plotted for each treatment group. * designates p < 0.05.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against phospho-ERK1/2 and total ERK1/2 (Cell Signaling; Beverly, MA), rabbit polyclonal antibodies against CXCR4 (Abcam, Cambridge, MA) and CXCR7 (Abcam and R&D Systems), a goat polyclonal antibody against β-arrestin-2 (Abcam), and a mouse monoclonal antibody against K-Ras (Calbiochem; San Diego, CA).

Techniques: Activity Assay, Pull Down Assay, Transfection, Control

Journal: Journal of Translational Medicine

Article Title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

doi: 10.1186/1479-5876-10-68

Figure Lengend Snippet: β-arrestin-2's role in CXCR4 and CXCR7-driven ERK phosphorylation . PANC-1 cells were transfected with control or β-arrestin-2 siRNA (100 nM) using RNAiMAX (10 μl). Cells were serum-starved and then exposed to CXCL12 (100 ng/ml) or CXCL11 (200 ng/ml) for 15 min. The immunoblot shows β-arrestin-2 and phospho-ERK1/2 expression with total ERK1/2 and GAPDH serving as loading controls.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against phospho-ERK1/2 and total ERK1/2 (Cell Signaling; Beverly, MA), rabbit polyclonal antibodies against CXCR4 (Abcam, Cambridge, MA) and CXCR7 (Abcam and R&D Systems), a goat polyclonal antibody against β-arrestin-2 (Abcam), and a mouse monoclonal antibody against K-Ras (Calbiochem; San Diego, CA).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

doi: 10.1186/1479-5876-10-68

Figure Lengend Snippet: The effect of CXCL12 on proliferation in pancreatic cancer cells . A quantitative ATP-based proliferation assay was performed on PANC-1 cells a) treated with CXCL12 and CXCL11 for 72 h.. b) PANC-1 and c) FG cells were transfected with control, CXCR4, or CXCR7 siRNA (100 nM) prior to 72 h of exposure with CXCL12 (200 ng/ml). Proliferation levels presented were normalized to each untreated baseline level, relative increases are depicted. The mean absorbance ± one SD was plotted for each treatment group. * designates p < 0.05.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies against phospho-ERK1/2 and total ERK1/2 (Cell Signaling; Beverly, MA), rabbit polyclonal antibodies against CXCR4 (Abcam, Cambridge, MA) and CXCR7 (Abcam and R&D Systems), a goat polyclonal antibody against β-arrestin-2 (Abcam), and a mouse monoclonal antibody against K-Ras (Calbiochem; San Diego, CA).

Techniques: ATP Proliferation Assay, Transfection, Control