integrin β1 shrna  (Santa Cruz Biotechnology)

Journal: Cellular & molecular biology letters

Article Title: Sialylation on vesicular integrin β1 determined endocytic entry of small extracellular vesicles into recipient cells.

doi: 10.1186/s11658-024-00562-0

Figure Lengend Snippet: Fig. 3 Identification of sialylated integrin β1 on sEV. A Identification of integrin β1 on YTS-1 sEV by MS. B YTS-1 sEV were purified by density gradient centrifugation. Levels of two sEV markers (TSG101, CD63) and integrin β1 in fractions 1–12 were analyzed by western blotting, and sialic acid levels were analyzed by lectin blotting. C Integrin β1 on sEV was enriched by IP and sialylation of vesicular integrin β1 was analyzed by lectin blotting. D Integrin β1 knockdown in YTS-1 (termed shA/B/C). shC (termed Y-shβ1) was used for further assay. GAPDH was used as loading control. E Uptake of ExoTracker-labeled sEV from Y-vec, Y-shβ1, and Y/ GPI-NEU1. F Y-vec sEV and Y-shβ1 sEV were treated with/ without sialidase and ExoTracker-labeled, and their uptake by HCV29 was analyzed by flow cytometry. G–I HCV29 was treated with Y-vec sEV or Y-shβ1 sEV, and proliferation (G), apoptosis (H), and migratory ability (I) were determined. J Effects of integrin β1 blockage on sEV uptake. K–M HCV29 was incubated with YTS-1 sEV pre-treated with IgG or integrinβ1 neutralizing antibodies, and proliferation (K), apoptosis (L), and migratory ability (M) were determined. N Levels of sialylated integrin β1 on sEV from plasma, determined by ELISA. O Levels of total integrin β1 in plasma determined by ELISA. P Levels of sialylated integrin β1 in plasma determined by ELISA

Article Snippet: Chemically synthesized oligonucleotides encoding integrin β1 short hairpin RNA (shRNA) were inserted in lentiviral plasmid Tet-pLKO-puro (Addgene plasmid #21915), and transfected into YTS-1 (termed Y-shβ1).

Techniques: Purification, Gradient Centrifugation, Western Blot, Knockdown, Control, Labeling, Flow Cytometry, Incubation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cellular & molecular biology letters

Article Title: Sialylation on vesicular integrin β1 determined endocytic entry of small extracellular vesicles into recipient cells.

doi: 10.1186/s11658-024-00562-0

Figure Lengend Snippet: Fig. 4 Effects of integrin β1 sialylation on sEV uptake. A Potential N-glycosylation sites (indicated by triangles) on integrin β1. Combined mutation to Asp of N-glycosylation sites 4–6 and 7–8 gave rise to Δ4–6 and Δ7–8. B Expression of WT integrin β1, Δ4–6, and Δ7–8 mutants. Tubulin was used as loading control. C Sialylation levels on integrin β1 in mutants by co-IP and western blotting. D Effects of mutant integrin β1 on sEV uptake. E–G HCV29 was treated with sEV from Y-vec, WT, and Δ7–8 mutant. Migratory ability (E), proliferation (F) and apoptosis (G) were determined. H Uptake of YTS-1 sEV in the presence of cilengitide (“RGD”). I Integrin β1/ FN interaction, assayed by co-IP and western blotting. J The interaction between integrin α5β1 and FN in cells expressing wild type integrin β1 and Δ7–8 mutant, assayed by co-IP and western blotting. K Integrin β1 and FN expression in sEV from Y-vec and Y-shβ1. TSG101 was used as loading control. L Integrin β1, α5 and FN levels in sEV from Y-vec and Y-shβ1. TSG101 was used as loading control

Article Snippet: Chemically synthesized oligonucleotides encoding integrin β1 short hairpin RNA (shRNA) were inserted in lentiviral plasmid Tet-pLKO-puro (Addgene plasmid #21915), and transfected into YTS-1 (termed Y-shβ1).

Techniques: Glycoproteomics, Mutagenesis, Expressing, Control, Co-Immunoprecipitation Assay, Western Blot

Journal: Cellular & molecular biology letters

Article Title: Sialylation on vesicular integrin β1 determined endocytic entry of small extracellular vesicles into recipient cells.

doi: 10.1186/s11658-024-00562-0

Figure Lengend Snippet: Fig. 5 Effects of integrin β1 sialylation on the pro-metastatic function of sEV. A Experimental workflow (schematic). B–E Representative photographs and haematoxylin and eosin (H&E) staining of livers or lungs. F, G Flag-tagged WT or Δ7–8 mutant integrin β1 expression in liver (F) or lung (G) by immunohistochemistry. H Distribution of sEV in mice after injection of sEV by tail vein

Article Snippet: Chemically synthesized oligonucleotides encoding integrin β1 short hairpin RNA (shRNA) were inserted in lentiviral plasmid Tet-pLKO-puro (Addgene plasmid #21915), and transfected into YTS-1 (termed Y-shβ1).

Techniques: Staining, Mutagenesis, Expressing, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: Control cell migration by engineering integrin ligand assembly

doi: 10.1038/s41467-022-32686-2

Figure Lengend Snippet: a Schematic illustration of precise control of integrin ligand presentation on nanofilaments via peptide assembly. b TEM images of nanofilaments obtained via molecular self-assembly and co-assembly of FFFIKLLI (100 μM) and FFF at various ratios, and the estimated molecular packing structures. IKLLI motif is presented in blue and FFF motif is presented in pink. The scale bars represent 200 nm. Three independent experiments were performed. c Zoom-in SEM images (false color) of HuH-7 cell edge and apical membrane after 3-day incubations. FFFIKLLI was maintained at a concentration of 100 μM. The cell body is highlighted in pink, while the nanofilaments are highlighted in blue. The scale bars represent 300 nm.

Article Snippet: Integrin β1 shRNA plasmid (#sc-29375-SH), Integrin α3 shRNA plasmid (#sc-35684-SH), Integrin α6 shRNA plasmid (#sc-43129-SH), and control shRNA plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology for knockdown of the target integrins.

Techniques: Control, Membrane, Concentration Assay

Journal: Nature Communications

Article Title: Control cell migration by engineering integrin ligand assembly

doi: 10.1038/s41467-022-32686-2

Figure Lengend Snippet: a Time-lapse series showing actin cytoskeleton (grey) and paxillin (green) in HuH-7 cells expressing mRuby-Lifeact-7 and mGFP-paxillin upon the treatment of FFFIKLLI (100 μM) for 12 hr. Scale bars represent 2 μm. Three independent experiments were performed. b F-actin phalloidin staining (magenta), integrin β1 (cyan) and paxillin (yellow) immunofluorescence in HuH-7 cell after 12 h treatment of FFFIKLLI (100 μM). Scale bar represents 2 μm. c Fluorescence intensity distribution profile of integrin β1, paxillin, and actin cytoskeleton along the yellow line on merged image of b . d 12 h time course Rac1 activity of HuH-7 cells with or without the treatment of FFFIKLLI (100 μM). Rac1 activity was measured by FRET. n = 6 cells (Ctrl) or 5 cells (FFFIKLLI). Symbols represent the mean FRET/CFP emission ratio ± s.d. e Representative FRET/CFP ratio images of HuH-7 cells expressing RaichuEV-Rac1 with or without the treatment of FFFIKLLI (100 μM) at the indicated time points and coded according to a pseudo-color scale, which ranges from yellow to purple with an increase in Rac1 activity. Scale bars represent 20 μm. Source numerical data are available in source data.

Article Snippet: Integrin β1 shRNA plasmid (#sc-29375-SH), Integrin α3 shRNA plasmid (#sc-35684-SH), Integrin α6 shRNA plasmid (#sc-43129-SH), and control shRNA plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology for knockdown of the target integrins.

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence, Activity Assay