functional blocking antibodies for integrin α 5 β 1 (jbs5)  (Merck KGaA)

Journal: Oncogene

Article Title: C1GALT1 high expression is associated with poor survival of patients with pancreatic ductal adenocarcinoma and promotes cell invasiveness through integrin α v

doi: 10.1038/s41388-020-01594-4

Figure Lengend Snippet: A Effects of C1GALT1 knockdown on cell spreading in HAPF-II and HPAC cells. Pancreatic cancer cells were treated with control (siControl) or C1GALT1 siRNA (siC1GALT1-3). Representative images showing amplified cells in one fourth of a field. Scale bars, 20 μm. Spreading cells were quantified under a phase-contrast microscope and are shown in the lower panel. * p < 0.05 by student’s t test. B C1GALT1 knockdown inhibited cell-extracellular matrix (ECM) adhesion. Cells were plated onto 96-well plates coated with 2.5 μg/μL of bovine serum albumin (BSA), collagen I (Col I), collagen IV (Col IV), fibronectin (FN), or laminin (LAM). Specific ECM-adhered cells were calculated by subtracting BSA-adhered cells. Results are presented as mean ± SD of six independent experiments. ** p < 0.01; *** p < 0.001 by student’s t test. C Effects of C1GALT1 knockdown on tyrosine phosphorylation of FAK. HPAF-II and HPAC cells were plated onto culture plates coated with 1 µg/mL of different ECM proteins, as indicated, in serum-free DMEM for 3 h. Changes in FAK phosphorylation at Y397 and Y925 were analyzed by Western blotting. GAPDH was used as an internal loading control. D Effects of C1GALT1 knockdown on Tn antigen expression of selected integrins, including α v , α 5 , β 1 , α 2 , and α 3. Changes in Tn expression were analyzed using VVA pull-down (PD) assays in C1GALT1 knockdown HAPF-II and HPAC cells. Proteins were detected by Western blot (WB) analysis. GAPDH was used as an internal loading control.

Article Snippet: Functional blocking antibodies for integrin β 1 (P4C10), integrin α 5 β 1 (JBS5), integrin α 5 (P1D6), and integrin α v (AV1) were purchased from Merck KGaA.

Techniques: Amplification, Microscopy, Western Blot, Expressing

Journal: Oncogene

Article Title: C1GALT1 high expression is associated with poor survival of patients with pancreatic ductal adenocarcinoma and promotes cell invasiveness through integrin α v

doi: 10.1038/s41388-020-01594-4

Figure Lengend Snippet: A Western blots showing C1GALT1 stable knockdown in HPAF-II and HPAC cells and C1GALT1 overexpression in MIAPaca2 cells. PDAC cells were transfected using lentivirus-mediated C1GALT1 shRNA in pLKO.1 vector (shC1GALT1) compared with its empty vector (shControl) and C1GALT1 was overexpressed using C1GALT1/pcDNA3.1 plasmid (C1GALT1) compared with its empty plasmid (mock). GAPDH was used as an internal loading control. B C1GALT1 knockdown or overexpression did not alter the expression of surface integrins β 1 , α 5 , α 5 β 1 , and α v in HAPF-II and HPAC cells analyzed by flow cytometry. Unstained cells were used as a negative control (-). C Effects of functional blocking antibodies against integrins on PDAC cell invasion. C1GALT1 knockdown HPAF-II and HPAC cells and C1GALT1 overexpressing MIAPaca2 cells were subjected to Matrigel invasion assays. Cells were treated with 10 μg/mL of blocking antibody, as indicated. IgG was used as a control. Invasion of HPAF-II, HPAC, and MIAPaca2 cells was analyzed after 24 and 48 h. Results are presented as mean ± SD of four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 by student’s t test. D Effects of functional blocking antibodies on FAK phosphorylation in PDAC cells using Western blotting. Functional blocking antibody against integrin α v or α 5 was used, as indicated, to treat HPAF-II and HPAC cells for 30 min before seeding to culture plates. IgG was used as a control. GAPDH was used as an internal loading control. E Western blots showing changes in Tn antigens on cell surface integrins α V and α 5 . Cells were surface biotinylated. Plasma Membrane Protein Extraction Kit (Abcam) was used for extraction and purification of plasma membrane proteins from HPAF-II and HPAC cells. VVA pull-down (PD) assays were performed to assess changes in Tn antigens on integrins. Proteins were detected by Western blot (WB) analysis. F A schematic diagram illustrating the proposed mechanism by which C1GALT1 promotes tumor growth and metastasis in pancreatic cancer. C1GALT1 modifies O-glycans on integrins, including α 5 , α v , and β 1 , which leads to altered integrin-FAK signaling. Integrin α v (red color) is proposed to play a critical role in C1GALT1-mediated invasiveness. This pathway is coordinated with other C1GALT1-regulated pathways, such as receptor tyrosine kinases (RTKs) and mucins, to promote tumor growth and metastasis in pancreatic cancer. FN fibronectin, VN vitronectin.

Article Snippet: Functional blocking antibodies for integrin β 1 (P4C10), integrin α 5 β 1 (JBS5), integrin α 5 (P1D6), and integrin α v (AV1) were purchased from Merck KGaA.

Techniques: Western Blot, Over Expression, Transfection, shRNA, Plasmid Preparation, Expressing, Flow Cytometry, Negative Control, Functional Assay, Blocking Assay, Protein Extraction, Purification