Journal: American Journal of Cancer Research

Article Title: Linc00662 m 6 A promotes the progression and metastasis of pancreatic cancer by activating focal adhesion through the GTF2B-ITGA1-FAK pathway

doi:

Figure Lengend Snippet: The FAK inhibitor-Y15 repressed the malignant progression of PC. A. Linc00662-overexpressing PC cells were treated with Y15 at different concentrations to identify the suitable concentrations. The results of western blotting showed that 2 µM and 5 µM of Y15 were the suitable concentrations in BxPC-3 cells and PANC-1 cells, respectively, which inhibited FAK autophosphorylation activity without effect on FAK expression. B. Western blotting showed that Y15 treatment dramatically repressed the levels of p-FAK, p-Paxillin, and p-Erk in PC cells. C. The CCK8 assay showed that Y15 treatment significantly reversed the increased proliferative capacity of Linc00662-overexpressing PC cells. D. The wound healing assay indicated that Y15 treatment significantly reversed the increased migratory capacity of Linc00662-overexpressing PC cells, magnification: 100 ×. E. The transwell assay indicated that Y15 treatment significantly reversed the increased invasive capacity of Linc00662-overexpressing PC cells, magnification: 100 ×. F. A mouse xenograft model was used to evaluate the effect of Y15 on cell growth in vivo. Images of transplanted subcutaneous tumors were displayed. G. Y15 treatment dramatically repressed tumor volume in mice bearing Linc00662-overexpressed PC cells. *: P < 0.05. H. Y15 treatment dramatically repressed subcutaneous tumor weight in mice bearing Linc00662-overexpressed PC cells. *: P < 0.05. Abbreviations: PC: pancreatic cancer, CCK8: cell counting kit-8.

Article Snippet: After 1 week, pre-established tumor xenografts were treated with Y15 (0.5 mg/kg, 2 ×/week × 3) (MCE, New Jersey, China).

Techniques: Western Blot, Activity Assay, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Assay, In Vivo, Cell Counting

Journal: American Journal of Cancer Research

Article Title: Linc00662 m 6 A promotes the progression and metastasis of pancreatic cancer by activating focal adhesion through the GTF2B-ITGA1-FAK pathway

doi:

Figure Lengend Snippet: Schematic model on the role of Linc00662 in regulating PC malignant progression through the GTF2B-ITGA1-FAK pathway. This study proposes a novel regulatory mechanism of Linc00662 in oncogene activation in PC. Linc00662 activates the transcription of ITGA1 by recruiting GTF2B in a m6A-dependent manner and promotes PC cell proliferation, invasion, and migration, which partly relies on the activation of focal adhesions through the ITGA1-FAK-Erk pathway. And Y15 treatment inhibits autophosphorylation of FAK and obviously represses tumor progression of Linc00662-overexpressing PC cells. Abbreviations: PC: pancreatic cancer, GTF2B: general transcription factor II-B, ITGA1: integrin alpha-1, m6A: N6-methyladenosine.

Article Snippet: After 1 week, pre-established tumor xenografts were treated with Y15 (0.5 mg/kg, 2 ×/week × 3) (MCE, New Jersey, China).

Techniques: Activation Assay, Migration

Journal: International Journal of Biological Sciences

Article Title: CPNE8 Promotes Gastric Cancer Metastasis by Modulating Focal Adhesion Pathway and Tumor Microenvironment

doi: 10.7150/ijbs.76425

Figure Lengend Snippet: CPNE8 acted on the Focal adhesion pathway to promote GC metastasis. ( A ) KEGG functional enrichment analysis revealed a significant correlation between CPNE8 expression and Focal adhesion pathway in AGS-sh- CPNE8 cells compared to the control cells. ( B ) There are relative changes of FAK (Gene Symbol as PTK2) and ERK mRNA levels in GC cells with knockdown or overexpression of CPNE8 . ( C ) Western blot analysis of FAK , p- FAK , total Erk1/2, and p-ERK protein expression in GC cells with knockdown or overexpression of CPNE8 . Relative protein expression (interested protein/ GAPDH ) was also quantified in GC cells. (D) EDU staining assay showed the proliferating cells with or without diminishing FAK expression, such as GSK2256098 treatment and knockdown of FAK . Photos were captured using a fluorescence microscope (Leica-Microsystems). DNA (blue) was stained with Hoechst. Cyan cells showed EDU/Hoechst-positive cells. The column diagram represented the proliferation rates in various GC cells. Data are presented as mean ± SD for at least three independent experiments. ( E ) Wound-healing assays revealed that both GSK2256098 and si -FAK could quantitatively reduce the CPNE8 -induced migration in BGC823 cells using Image J software. ( F ) Transwell assays confirmed that GSK2256098 or knocking down FAK could inhibit migration and invasiveness of GC cells with overexpression of CPNE8 . ( G ) The representative images showed the adhesion ability of GC cells with knockdown or overexpression of CPNE8 compared to the control cells and the attenuation of BGC823-oe- CPNE8 with GSK2256098 or knockdown of FAK . The number of GC cells shown in the bar graph that adhered to the plates coated after 2h of incubation was quantified using Image J software.

Article Snippet: The medium was then replaced with fresh medium containing 0-10 μM GSK2256098 (TargetMol, Cat. #T2281), a small-molecule FAK inhibitor.

Techniques: Functional Assay, Expressing, Over Expression, Western Blot, Staining, Fluorescence, Microscopy, Migration, Software, Incubation

Journal: Journal of Cell Science

Article Title: Distinct focal adhesion protein modules control different aspects of mechanotransduction

doi: 10.1242/jcs.195362

Figure Lengend Snippet: FAK and paxillin phosphorylation are involved in mechanotransduction. (A,B) Graphs showing the percentage of the cell area that is composed of pY118-paxillin- or pY397-FAK-positive FAs (A) and the ratio of pY118-paxillin or pY397-FAK to total paxillin (B) in cells plated on the indicated substrates. Data are pooled from three independent repeats, n=43–50 cells. ***P<0.001 (one-way ANOVA with Tukey post-hoc test). (C) Fluorescence loss curves (mean±s.e.m.) of vinculin and tensin FLAP experiments performed in NIH3T3 cells plated on the indicated substrates and treated with FAKi+Srci. (D) t½ FLAP graph showing that the turnover rates of vinculin and tensin have different responses to substrate stiffness following treatment with FAKi and Srci. Data in C and D are pooled from three independent repeats, n=58–75 FAs. *P<0.05; ***P<0.001; n.s., no significant difference (one-way ANOVA with Tukey post-hoc test). For the box plots in A,B,D, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range of values.

Article Snippet: The FAK inhibitor (FAKi; AZ13256675) and Src inhibitor (Srci; AZD0530) were obtained from Astra Zeneca, and are available from the pharmacology toolbox ( http://openinnovation.astrazeneca.com/what-we-offer/pharmacology-toolbox/ ).

Techniques: Phospho-proteomics, Fluorescence

Journal: Journal of Cell Science

Article Title: Distinct focal adhesion protein modules control different aspects of mechanotransduction

doi: 10.1242/jcs.195362

Figure Lengend Snippet: FAK and Src kinase activity influence FX formation and turnover. (A) Representative images showing NIH3T3 cells transfected with GFP–paxillin and treated with DMSO, FAKi or FAKi+Srci. Scale bar: 10 μm. Red boxes indicate the regions that are shown as a magnified view (below). Scale bars: 5 μm. (B) Graph showing the adhesion size in NIH3T3 cells under the indicated conditions. Data are pooled from three independent repeats, n=62–68 cells. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range of values. ***P<0.001 (Student's t-test). (C) Representative time-lapse images of DMSO or drug-treated NIH3T3 cells transfected with GFP–paxillin. The colored images show adhesions present at the indicated time points. Overlap in color shows that the GFP–paxillin-containing FA has persisted between the time points (yellow, 0 and 30 min; turquoise, 30 and 60 min; white, all three time points). Note the persistence of FAs following FAKi and Srci treatment. (D) Representative images (above) and zoomed regions (below) of NIH3T3 cells pre-treated in suspension with the indicated drugs and co-stained for paxillin and pY118-paxillin after plating on FN-coated coverslips. Graphs show the fluorescence intensity across the indicated line. Note the presence of paxillin-positive FXs even in the absence of intracellular tension and tyrosine phosphorylation. Scale bar: 10 μm (upper images) and 5 μm (lower images).

Article Snippet: The FAK inhibitor (FAKi; AZ13256675) and Src inhibitor (Srci; AZD0530) were obtained from Astra Zeneca, and are available from the pharmacology toolbox ( http://openinnovation.astrazeneca.com/what-we-offer/pharmacology-toolbox/ ).

Techniques: Activity Assay, Transfection, Suspension, Staining, Fluorescence, Phospho-proteomics

Journal: Journal of Cell Science

Article Title: Distinct focal adhesion protein modules control different aspects of mechanotransduction

doi: 10.1242/jcs.195362

Figure Lengend Snippet: FAK and Src kinase activity are required for lamellipodial protrusions, cell migration and spreading. (A) Representative recordings following the indicated drug treatments of NIH3T3 cells transfected with RFP-lifeact. The color-coded outlines show the position of the cell during the course of the 90 min movie (4 min intervals). Kymographs (below) were taken at the indicated position (red line). Note the dramatic drop in protrusive behavior following the inhibition of FAK and Src. Scale bar: 10 μm. (B) Quantification of the number of filopodia around the cell periphery following treatment with DMSO, FAKi or FAKi+Srci. Graph shows the mean stress of all cells analyzed (scatter dots), n=31–35 cells. **P<0.01 (one-way ANOVA with Tukey post-hoc test). (C) Representative trajectories of cells recorded for 16 h on 10 μm-wide fibronectin stripes; images of NIH3T3 cells plated on fibronectin stripes (inset). Note the decreased migration rate of cells treated with FAKi and FAKi+Srci. (D) Graph showing the increase in cell area over time following plating on FN-coated cover slips. Error bars are ±s.e.m., n=113–233 cells. *P<0.05; **P<0.01; ***P<0.001; n.s., no significant difference (one-way ANOVA with Tukey post-hoc test). (E) Representative cell images and stress maps of traction force microscopy. Stress maps are colored to show intensity. Scale bar: 10 μm. Graph shows the mean stress of each cell analyzed (scatter dots), n=34–45 cells. n.s., no significance (one-way ANOVA with Tukey post-hoc test). For the box plots in B and E, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range of values.

Article Snippet: The FAK inhibitor (FAKi; AZ13256675) and Src inhibitor (Srci; AZD0530) were obtained from Astra Zeneca, and are available from the pharmacology toolbox ( http://openinnovation.astrazeneca.com/what-we-offer/pharmacology-toolbox/ ).

Techniques: Activity Assay, Migration, Transfection, Inhibition, Microscopy