αv p3g8  (Developmental Studies Hybridoma Bank)

Journal: eLife

Article Title: Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin

doi: 10.7554/eLife.69028

Figure Lengend Snippet: ( A ) Schematic for testing monoclonal antibodies to block integrin β1 (P5D2), α5 (P1D6), αV (P3G8), and α4 (P4G9). ( B ) cTnT + cells measured by flow cytometry at 15 days differentiation when anti-human integrin β1 antibody was added on day –2 or day 0 at 3 µg/ml in the matrix sandwich protocol as shown in A. ( C ) cTnT + cells measured by flow cytometry at 15 days differentiation when anti-human integrin β1 antibody was added on day 0 at concentrations of 1, 2.5, and 5 µg/ml in the matrix sandwich protocol as shown in A. ( D ) cTnT + cells measured by flow cytometry at 15 days differentiation when anti-human integrin α5, αV or α4 antibody was added on day –2 or day 0 at 3 µg/ml in the matrix sandwich protocol as shown in A. ( E ) cTnT + cells measured by flow cytometry at 15 days differentiation when anti-human integrin α4 antibody was added on day 0 at concentrations of 1, 2.5, and 5 µg/ml in the matrix sandwich protocol. ( F ) cTnT + cells measured by flow cytometry at 15 days differentiation when anti-human integrin αV antibody was added on day 0 at concentrations of 1, 2.5, and 5 µg/ml in the matrix sandwich protocol. The % of cTnT + cells in each group was normalized to the control group to combine replicates from multiple differentiations and to compare across different antibodies blocking. N≥3 biological replicates. Data are from DF19-9-11T iPSC line. Error bars represent SEM. *p<0.05, one-way ANOVA with post-hoc Bonferroni test.

Article Snippet: Blocking antibodies, P5D2 (anti- human integrin β1), P1D6 (anti-human integrin α5), P3G8 (anti-human integrin αV) and P4G9 (anti-human integrin α4) (all from Developmental Studies Hybridoma Bank), and ILK inhibitor cpd22 (EMD Millipore) were diluted in the media to make the final concentrations and added to the culture at the indicated time points.

Techniques: Bioprocessing, Blocking Assay, Flow Cytometry, Control

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: Antibodies against integrin α v (P3G8), integrin α 2 (CD49b), and integrin α 3 (CD49c) were obtained from Merck KGaA (Darmstadt, Germany).

Techniques: Western Blot, Knockdown, Over Expression, Transfection, shRNA, Plasmid Preparation, Control, Expressing, Flow Cytometry, Negative Control, Functional Assay, Blocking Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Protein Extraction, Extraction, Purification