Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: Integrin α5 and β1 are associated with pancreatic cancer patient survival. Integrin β1 and α5 mRNA levels in primary tumors were compared with normal tissue using TCGA data. ( A ) Primary tumors have higher gene expression of integrin β1 than normal tissues (# p < 0.0001, n = 165 for normal and n = 178 for tumors) red lines indicate the mean and SD of the values. ( B ) Increased expression of integrin β1 (red lines) associates with lower survival in patients as depicted by KM plot p < 0.001. ( C ) Integrin α5 also has higher gene expression than normal tissues (# p < 0.0001, n = 165 for normal and n = 183 for tumors). ( D ) Integrin α5 did not show a clear relationship with patient survival p > 0.05.

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: The deletion of the β1 integrin gene from MIA PaCa-2 cells decreases tumor growth on the 3D matrix. ( A ) Tumors formed by MIA PaCa-2 cells expressing the integrin β1 gene and cells where the integrin β1 gene removed were stained for F-Actin (phalloidin, green) to estimate the size of the tumor. Scale bar 20 µm. ( B ) 3D projection of z stack indicate the tumor size. ( C ) The projection of the intensity of actin staining (view 2.5D) and (D) Tumor volumes were measured after the acquisition of z-stack slices with confocal microscope. These images were processed using ImageJ software to determine the volume. One-way ANOVA was performed with Bonferroni’s test to compare between groups. * indicates a statistically significant difference between WT and Clone 2, n = 12, p < 0.001). # indicates the statistically significant difference between groups WT and Clone 4 n = 12, p < 0.001. Data shown are mean ± SEM, unit: µm 3 .

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Staining, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: Integrin β1 deletion decreased the cellular adhesion of MIA PaCa-2 cells to vitronectin and fibronectin. ( A ) Integrin β1 expressing MIA PaCa-2 and integrin β1 KO clones were immunoblotted by anti-integrin β1 antibody. GAPDH was used as the loading control. ( B ) Cell lysate was immunoblotted with integrin α5 antibodies. GAPDH was used as the loading control. (C) Quantification of integrin α5 expression normalized to GAPDH, mean ± SEM from three independent experiments ( n = 3). One-way ANOVA performed with Bonferroni’s test indicated a statistically significant decrease in cell adhesion, * p < 0.001 between WT and clone2, # p < 0.001 between WT and clone4. ( D ) The expression of integrin α5 mRNA by RT-PCR. ΔΔCt values normalized with to 18s n = 3, * p < 0.001 between WT and clone2, # p < 0.001 between WT and clone4. ( E ) Percentage change in cellular adherence of integrin β1 KO to fibronectin (5 µg/mL) compared to the integrin β1 expressing cells, n = 3, * p < 0.01 between WT and clone2, # p < 0.01 between WT and clone4. ( F ) Comparison of cellular adhesion between integrin β1 expressing MIA PaCa-2 cells and integrin β1 KO cells to vitronectin (5 µg/mL) n = 3, * p < 0.01 between WT and clone2, # p < 0.01 between WT and clone4.

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Clone Assay, Control, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: MIA PaCa-2 integrin β1 KO cells have decreased spreading ability compared to integrin β1 expressing cells. Cells were plated on either fibronectin or vitronectin coated plates for 4 h and the cells were stained with actin. ( A ) Actin staining of cell spread on 5 µg/mL fibronectin visualized by immunostaining (F-actin, phalloidin, green; and nucleus, DAPI, blue). Scale bar 20 µm. ( B ) Actin staining of cell spread on vitronectin visualized by immunostaining (Green: F-actin using phalloidin, and Blue: nucleus DAPI). Scale bar 20 µm. ( C , D ) Quantitative assessment of cell spreading in fibronectin (area: µm 2 ). One-way ANOVA was performed with Bonferroni’s post-hoc test to compare between groups. * Indicates a statistically significant difference between groups (integrin β1 expressing MIA PaCa-2 and integrin β1 KO Clone 2) ( n = 40, p < 0.001). # Shows a statistically significant difference between groups (integrin β1 expressing MIA PaCa-2 and integrin β1 KO Clone 4) ( n = 40, p < 0.001). Data shown are mean ± SEM, unit: µm 2 .

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: Impaired focal adhesion contributes to less cell spreading in MIA PaCa-2 integrin β1 KO cells ( A ) Phospho-paxillin immunostaining shows the decrease in the phosphorylation status of paxillin in MIA PaCa-2 and integrin β1 KO cells. Visualized by immunostaining F-actin, phalloidin, green, p-paxillin, Alexa flour 555, red). Scale bar 20 µm. ( B ) Immunoblotting confirmed the downregulation of p-paxillin. Normalized p-paxillin expression with GAPDH shows a significant decrease in expression of p-paxillin. ( C ) No significant change in paxillin expression normalized with GAPDH between WT and clones. One-way ANOVA was performed by Bonferroni’s test to compare between groups ( n = 3). * indicates a statistically significant difference between groups (integrin β1 expressing MIA PaCa-2 and integrin β1 KO cells Clone 2, p < 0.001). # Shows a statistically significant difference between groups (integrin β1 expressing MIA PaCa-2 and integrin β1 KO cells Clone 4, p < 0.001).

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Immunostaining, Phospho-proteomics, Western Blot, Expressing, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: Integrin β1 deletion downregulated Kindlin-2 and TGFβ receptor 2 expression. ( A ) Cell lysate (30 µg/lane) from integrin β1 expressing MIA PaCa-2 and integrin β1 KO clones are immunoblotted for kindlin-2. Normalized protein expression with GAPDH shows a decrease in kindlin-2 expression in clones compared to the normal MIA PaCa-2 cells. ( B ) The mRNA expression quantified by RT-PCR analysis shows a significant decrease in the expression of kindlin-2 by the deletion of integrin β1. ( C ) Lysates were immune blotted with antibody recognizing TGFβ receptor 2. GAPDH is used as the loading control. Normalized protein expression shows downregulation of TGFβ receptor 2. ( D ) The mRNA expression quantified by RT-PCR analysis shows a significant decrease in the expression of TGFβ receptor 2 by the deletion of integrin β1. One-way ANOVA was performed by Bonferroni’s test to compare integrin β1 expressing MIA PaCa-2 and integrin β1 KO clones indicate a statistically significant difference n = 3, * WT and Clone 2 p < 0.001; # WT and Clone 4 p < 0.001. Quantification is expressed as mean ± SEM.

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

doi: 10.3390/ijms221910599

Figure Lengend Snippet: Integrin β1 deletion downregulated Smad2/3 phosphorylation. ( A ) Cell lysate from integrin β1 expressing MIA PaCa-2 and integrin β1 KO clones is immunoblotted for p-Smad2/3. ( A ) Immunoblotting of p-Smad2/3 shows inhibited downstream cell signaling pathways. Normalized P Smad-2/3 is shown using GAPDH as control. ( B ) Total expression of Smad-2/3 shows no significant change in the expression of Smad-2/3. One-way ANOVA performed with Bonferroni’s test to compare integrin β1 expressing MIA PaCa-2 and integrin β1 KO clones indicates a statistically significant difference n = 3, * WT and Clone 2 p < 0.001; # WT and Clone 4 p < 0.001. Quantification is expressed as mean ± SEM.

Article Snippet: Integrin β1-KO MIA, PaCa-2 cell lines were established using Integrin β1 CRISPR/Cas9 knock out Plasmids (sc-421171-NIC-2: Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Phospho-proteomics, Expressing, Clone Assay, Western Blot, Protein-Protein interactions, Control