αv p3g8  (Developmental Studies Hybridoma Bank)

Journal: Thoracic Cancer

Article Title: Epithelial‐Mesenchymal Plasticity in the D‐Meso‐Sonobe Mesothelioma Cell Line: A Putative Model of Epithelial–Mesenchymal Transition in Mesothelioma

doi: 10.1111/1759-7714.70091

Figure Lengend Snippet: Immunohistochemical staining of xenoplanted D‐Meso‐Sonobe tumor cells. Zeb1 immunoreactivity was barely found in xenoplanted tumor cells (a and b). Furthermore, nuclear Zeb1 immunoreactivity was found in various tumor cells at the cancer invasion front (indicated by ☆ in a and b). Cytoplasmic yes‐associated protein (YAP) immunoreactivity was found in center of the xenoplanted tumor (c), whereas nuclear YAP immunoreactivity was also found in tumor invasion front (d). Integrin Subunit Alpha V and Actin alpha 2 immunoreactivities were focally found in the xenoplanted tumor (e and f, respectively). LOXL1 immunoreactivity was found at the invasion front near muscle cells (indicated by white arrowhead), while minimum staining was observed in the non‐invasion front (indicated by black arrow). Scale bars represent 100 μm (a and g) and 50 μm (b–f).

Article Snippet: The rabbit antibodies against Integrin Subunit Alpha V (ITGAV) (cat. no. 27096‐1‐AP), and actin alpha 2 (ACTA2) (cat. no. 14395‐1‐AP) were purchased from Proteintech.

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Communications

Article Title: Galectin 3‐binding protein (LGALS3BP) depletion attenuates hepatic fibrosis by reducing transforming growth factor‐β1 (TGF‐β1) availability and inhibits hepatocarcinogenesis

doi: 10.1002/cac2.12600

Figure Lengend Snippet: Interaction of LGALS3BP with ITGαV and promotion of ITGαV accumulation on the cell membrane to release TGF‐β1. (A) Measurement of active TGF‐β1 and total TGF‐β1 in the culture medium of Hepa‐1c1c7 cells upon rLGALS3BP treatment. (B) Experimental outline: whole cell extracts of LGALS3BP ‐KI primary hepatocytes were immunoprecipitated with normal IgG or α‐myc. Immunoprecipitants were analyzed by LC‐MS/MS. (C) Protein‐protein interactions in the cluster 4 among the LGALS3BP‐binding protein networks. (D) Co‐immunoprecipitation assays to detect the interaction between secreted LGALS3BP and ITG proteins using anti‐myc antibody with whole cell extracts of empty vector or myc‐LGALS3BP transfected SNU449 cells. (E) ELISA for the detection of active TGF‐β1 in Hepa‐1c1c7, SNU447, and HepG2 cells in the culture medium upon rLGALS3BP ± GLPG0187 treatment. Data represented as mean ± SD, n = 3. * P < 0.05 and ** P < 0.05 (Two‐way ANOVA). (F) Representative images of PLA results using HepG2 cells. Empty vector or myc‐epitope tagged LGALS3BP was transiently transfected into HepG2 cells to detect its interaction with ITGαV. Red dots indicate the protein‐protein interactions. Blue dots denote DAPI nuclei staining. (G) Immunofluorescence staining of transfected myc‐LGALS3BP (Green) and ITGαV (Pink). (H) Immuno‐fluorescence staining of ITGαV and F‐actin in Hepa‐1c1c7 cells upon rLGALS3BP treatment for 1h. Quantification of total stained area of ITGαV and F‐actin were shown on the left with five scanned images per slides of three independent experiments. Data represented as mean ± SD, ** P < 0.01 and *** P < 0.001 (Student's t‐test). (I) Immunofluorescence staining of ITGαV and F‐actin in HepG2 cells upon rLGALS3BP treatment for 1h. Quantification of total stained area of ITGαV and F‐actin were shown on the left with five scanned images per slides of three independent experiments. Data represented as mean ± SD, * P < 0.05 and *** P < 0.001 (Student's t‐test). (J) JunB ChIP‐qPCR analysis of TGFB1 JUNB‐RE locus using Hepa‐1c1c7 cells upon rLGALS3BP ± GLPG0187 treatment. Data represented as mean ± SD, n = 3. *** P < 0.001 (Student's t ‐test). (K) Measurement of active TGF‐β1 in the culture medium of Hepa‐1c1c7 cells upon rLGALS3BP ± Defactinib treatment. Data represented as mean ± SD, n = 3. * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student's t ‐test). Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; F‐actin, filamentous actin; IP, immunoprecipitation; ITGαV, integrin subunit alpha V; JUNB‐RE, JunB response elements; LC‐MS/MS, Liquid Chromatography with tandem mass spectrometry; PH, primary hepatocytes; PLA, Proximity Ligation Assay; rLGALS3BP, recombinant LGALS3BP; TGFB1 , transforming growth factor beta 1; WCE, whole cell extract.

Article Snippet: Primary antibodies against phosphorylated Mothers against decapentaplegic homolog 2 (SMAD) family member 2 (p‐Smad2, #3108, 1:3,000, Cell signaling technology [CST], Danvers, MA, USA), Smad2 (#5339, 1:3,000, CST), JunB (#3753, 1:3,000, CST), β‐actin (#8457, 1:3,000, CST), Myc tag (M192‐3, 1:3,000, Medical & Biological Laboratories, Tokyo, Japan), Cyclophilin C‐associated protein (CyCAP)/mouse LGALS3BP (#28128, 1:1,000, Immuno‐Biological Lab, Japan, Fujioka, Japan), TGF‐β1 (ab215715, 1:3,000, Abcam), Integrin subunit alpha V (ITGaV, #60896, 1:3,000, CST), Phosphorylated focal adhesion kinase (FAK) (#3283, 1:3,000, CST), FAK (#3285, 1:3,000, CST) and vascular cell adhesion molecule 1 (VCAM1, ab134047, 1:3,000, Abcam).

Techniques: Membrane, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions, Binding Assay, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Fluorescence, ChIP-qPCR, Liquid Chromatography, Mass Spectrometry, Proximity Ligation Assay, Recombinant

Journal: Cancer Communications

Article Title: Galectin 3‐binding protein (LGALS3BP) depletion attenuates hepatic fibrosis by reducing transforming growth factor‐β1 (TGF‐β1) availability and inhibits hepatocarcinogenesis

doi: 10.1002/cac2.12600

Figure Lengend Snippet: JunB‐TGF‐β1 feedback loop initiation by LGALS3BP via the activation of ITGαV‐mediated rearrangement of F‐actin cytoskeleton. (A) Immunofluorescence staining of phosphorylated FAK and F‐actin in Hepa‐1c1c7 cells upon rLGALS3BP ± GLPG0187 treatment for 1h. Quantification of total stained area of p‐FAK and the length of elongated filamentous actin from the p‐FAK loci were shown on the left with five scanned images per slides of three independent experiments. Total numbers of F‐actin used in the measurement were n = 54, n = 120, n = 67, and n = 69 from the left column. Data represented as mean ± SD, *** P < 0.001 (Two‐way ANOVA). (B) qRT‐PCR analyses to measure the expression levels of JUNB and TGFB1 in Hepa‐1c1c7 cells upon rLGALS3BP ± GLPG0187 treatment. Data represented as mean ± SD, ** P < 0.01 and *** P < 0.001 (Two‐way ANOVA). (C) Western blottings to detect the expression levels of indicated proteins. Quantification of the band intensities were shown on the left. Data represented as mean ± SD, n = 3. *** P < 0.001 (Two‐way ANOVA). (D) Immunofluorescence staining of phosphorylated FAK and F‐actin in SNU449 cells upon rLGALS3BP ± GLPG0187 treatment for 1h. Quantification of total stained area of p‐FAK and the length of elongated filamentous actin from the p‐FAK loci were shown on the left with five scanned images per slides of three independent experiments. Total numbers of F‐actin used in the measurement were n = 41, n = 80, n = 30, and n = 35 from the left column. Data represented as mean ± SD, *** P < 0.001 (Two‐way ANOVA). (E) qRT‐PCR analyses to measure the expression levels of JUNB and TGFB1 in SNU449 cells upon rLGALS3BP ± GLPG0187 treatment. Data represented as mean ± SD, * P < 0.05, ** P < 0.01, and *** P < 0.001 (Two‐way ANOVA). (F) Western blots to detect the expression levels of indicated proteins. Quantification of the band intensities were shown on the left. Data represented as mean ± SD, n = 3. * P < 0.05, ** P < 0.01, and *** P < 0.001 (Two‐way ANOVA). Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; F‐actin, filamentous actin; F‐actin, filamentous actin; ITGαV, integrin subunit alpha V; p‐FAK, phosphorylated focal adhesion kinase; rLGALS3BP, recombinant LGALS3BP; TGFB1, transforming growth factor beta 1.

Article Snippet: Primary antibodies against phosphorylated Mothers against decapentaplegic homolog 2 (SMAD) family member 2 (p‐Smad2, #3108, 1:3,000, Cell signaling technology [CST], Danvers, MA, USA), Smad2 (#5339, 1:3,000, CST), JunB (#3753, 1:3,000, CST), β‐actin (#8457, 1:3,000, CST), Myc tag (M192‐3, 1:3,000, Medical & Biological Laboratories, Tokyo, Japan), Cyclophilin C‐associated protein (CyCAP)/mouse LGALS3BP (#28128, 1:1,000, Immuno‐Biological Lab, Japan, Fujioka, Japan), TGF‐β1 (ab215715, 1:3,000, Abcam), Integrin subunit alpha V (ITGaV, #60896, 1:3,000, CST), Phosphorylated focal adhesion kinase (FAK) (#3283, 1:3,000, CST), FAK (#3285, 1:3,000, CST) and vascular cell adhesion molecule 1 (VCAM1, ab134047, 1:3,000, Abcam).

Techniques: Activation Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Western Blot, Recombinant

Journal: Cancer Communications

Article Title: Galectin 3‐binding protein (LGALS3BP) depletion attenuates hepatic fibrosis by reducing transforming growth factor‐β1 (TGF‐β1) availability and inhibits hepatocarcinogenesis

doi: 10.1002/cac2.12600

Figure Lengend Snippet: Reduction of the steatohepatitis‐induced hepatocarcinogenesis by LGALS3BP depletion. (A) Experimental outline of the HCC mouse model. LGALS3BP +/+ and LGALS3BP −/− mice were injected intraperitoneally with 25 mg/kg DEN at 2 weeks after birth and fed an HFD for 26 weeks beginning at 6 weeks of age. All the mice were euthanized at 32 weeks of age. (B) Body weights of control and LGALS3BP ‐KO mice on the day of euthanasia ( n = 12‐13). (C) Tumor scores of the control and KO mouse livers. Tumors ≤ 5 mm were assigned a value of 1 and tumors > 5 mm were multiplied by 2. Tumor scores were determined as the sum of the values. Data represented as mean ± SD, n = 12‐13. *** P < 0.001 (Student's t ‐test). (D) ELISA assays for the measurement of active or total TGF‐β1 levels in the serum at the time of the sacrifice. Data represented as mean ± SD, n = 12‐13. ** P < 0.01 (Student's t ‐test) (E) The correlation between the tumor score and serum LGALS3BP levels in the control mice ( n = 12). (F) The correlation between the TGFB1 and LGALS3BP in the tumor tissues of control mice. (G) Upper panels: whole tumor‐bearing mouse livers and its H&E‐stained sections; lower panels: liver sections stained with H&E, Sirius Red, MT, and α‐SMA IHC. Quantification of the positive stained areas were shown on the left. The stained areas were shown as percentages of the total area of each liver sections. Data represented as mean ± SD, n = 10‐12. ** P < 0.01 and *** P < 0.001 (Student's t ‐test). (H) qRT‐PCR analysis of the indicated genes in hepatic tumors isolated from control and LGALS3BP ‐KO mice. Data represented as mean ± SD, n = 10‐12. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Student's t ‐test). (I) Representative western blottings to detect the expressions of indicated proteins in the cont. and KO mice liver tissues. (J) Representative immunohistochemical staining of TGF‐β1, JunB, and Ki‐67 in the indicated mouse liver tissues. Quantification of the positively stained area were shown below. The stained areas were shown as percentages of the total area of each liver sections. Data represented as mean ± SD, n = 16‐17 for TGF‐β1, n = 10‐11 for JunB and Ki‐67. * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student's t ‐test). Abbreviations: COL1A1 , collagen type I alpha 1; Cont., control; DEN, diethylnitrosamine; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; H&E, hematoxylin and eosin stain; H&E, hematoxylin and eosin stain; HFD, high fat diet; ITGaV, integrin subunit alpha V; KO, knockout; LGALS3BP , lectin galactoside‐binding soluble 3 binding protein; MT, Masson's trichrome stain; MT, Masson's trichrome stain; PDGFB , platelet derived growth factor subunit B; p‐FAK, phosphorylated focal adhesion kinase; SMA, alpha‐smooth muscle actin; TGFB1 , transforming growth factor beta 1; VCAM1 , vascular cell adhesion protein 1; α‐SMA, alpha‐smooth muscle actin.

Article Snippet: Primary antibodies against phosphorylated Mothers against decapentaplegic homolog 2 (SMAD) family member 2 (p‐Smad2, #3108, 1:3,000, Cell signaling technology [CST], Danvers, MA, USA), Smad2 (#5339, 1:3,000, CST), JunB (#3753, 1:3,000, CST), β‐actin (#8457, 1:3,000, CST), Myc tag (M192‐3, 1:3,000, Medical & Biological Laboratories, Tokyo, Japan), Cyclophilin C‐associated protein (CyCAP)/mouse LGALS3BP (#28128, 1:1,000, Immuno‐Biological Lab, Japan, Fujioka, Japan), TGF‐β1 (ab215715, 1:3,000, Abcam), Integrin subunit alpha V (ITGaV, #60896, 1:3,000, CST), Phosphorylated focal adhesion kinase (FAK) (#3283, 1:3,000, CST), FAK (#3285, 1:3,000, CST) and vascular cell adhesion molecule 1 (VCAM1, ab134047, 1:3,000, Abcam).

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR, Isolation, Western Blot, Immunohistochemical staining, H&E Stain, Knock-Out, Binding Assay, Derivative Assay

Journal: Journal of Experimental & Clinical Assisted Reproduction

Article Title: Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells

doi: 10.1186/1743-1050-2-4

Figure Lengend Snippet: Localisation of E-cadherin and integrin subunit α v . A-D : E-cadherin; E-H : integrin subunit α v , both detected by confocal laser scanning microscopy. Note that both adhesion molecules are located in all plasma membrane domains, including the apical one. Typical patterns are presented for apical (apical) and basal (basal) cell poles as well as for the middle part (mid) of cells. D,H: negative controls.

Article Snippet: Mouse monoclonal antibody against α v integrin subunit (0770) was obtained from Dianova (Hamburg, Germany).

Techniques: Confocal Laser Scanning Microscopy

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