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: Reproduction (Cambridge, England)

Article Title: OPN Binds Alpha V Integrin to Promote Endothelial Progenitor Cell Incorporation into Vasculature

doi: 10.1530/REP-19-0358

Figure Lengend Snippet: A) Integrin expression by pEPCs. RT-PCR analyses of integrin subunits expressed in pEPCs from passage 22. A band indicates the presence of a particular integrin subunit. B) Alpha v integrin on EPCs directly binds to OPN. Top panel: pEPCs were surface-labeled with biotin. detergent extracts of the cells mixed with OPN-Sepharose, integrins bound to OPN eluted with EDTA, and wash (W) and EDTA eluates (E1–E8) separated by SDS-PAGE. Bottom panel: Immunoprecipitation of pooled EDTA eluates (E2–E4) was performed using Protein A-Sepharose and antibodies directed to the indicated integrin subunits. Samples were separated by SDS-PAGE under non-reducing conditions. C) Immunofluorescence staining of pEPCs cultured on OPN using antisera directed to the αv integrin subunit shows aggregates of immunoreactive αv integrin at the base of the cells suggesting the assembly of focal adhesions (width of field = 140 μm).

Article Snippet: After washing with PBS containing 0.3% vol/vol Tween 20, cells were blocked with 10% vol/vol goat serum and incubated overnight at 4°C with 2 μg/μl rabbit polyclonal antibodies to von Willebrand factor (vWF; BD Biosciences, San Jose, CA), platelet endothelial cell adhesion molecule (PECAM-1; Santa Cruz, Dallas, Texas), vascular endothelial cadherin (VE-cadherin; Enzo Life Sciences, Farmingdale, NY), αv integrin subunit (AB1930; Chemicon, Temecula, CA), and mouse antibodies to focal adhesion kinase (FAK; BD Transduction Laboratories, Lexington, KY), vinculin (Sigma-Aldrich), and paxillin (Santa Cruz).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Labeling, SDS Page, Immunoprecipitation, Immunofluorescence, Staining, Cell Culture

Journal: Reproduction (Cambridge, England)

Article Title: OPN Binds Alpha V Integrin to Promote Endothelial Progenitor Cell Incorporation into Vasculature

doi: 10.1530/REP-19-0358

Figure Lengend Snippet: Porcine EPCs dose-dependently bind to recombinant rat OPN in an RGD-dependent manner. Adhesion assays were conducted with recombinant rat OPN containing an intact integrin binding sequence (RGD) or, rat OPN with a mutated integrin binding sequence (RAD). Porcine EPCs dose-dependently bound to recombinant rat OPN containing an RGD sequence, but not the mutated RAD control, indicating a dependence on the RGD sequence to promote pEPC attachment to OPN. Values represent average absorbance readings of adhered cells stained with Amido black (595 nm; 3 wells/data point).

Article Snippet: After washing with PBS containing 0.3% vol/vol Tween 20, cells were blocked with 10% vol/vol goat serum and incubated overnight at 4°C with 2 μg/μl rabbit polyclonal antibodies to von Willebrand factor (vWF; BD Biosciences, San Jose, CA), platelet endothelial cell adhesion molecule (PECAM-1; Santa Cruz, Dallas, Texas), vascular endothelial cadherin (VE-cadherin; Enzo Life Sciences, Farmingdale, NY), αv integrin subunit (AB1930; Chemicon, Temecula, CA), and mouse antibodies to focal adhesion kinase (FAK; BD Transduction Laboratories, Lexington, KY), vinculin (Sigma-Aldrich), and paxillin (Santa Cruz).

Techniques: Recombinant, Binding Assay, Sequencing, Staining

Journal: Reproduction (Cambridge, England)

Article Title: OPN Binds Alpha V Integrin to Promote Endothelial Progenitor Cell Incorporation into Vasculature

doi: 10.1530/REP-19-0358

Figure Lengend Snippet: Silencing of αv integrin in pEPCs using siRNA demonstrates that αv is required for pEPC attachment to OPN to form focal adhesions. A) Left panel: Western blots confirming successful knockdown of αv integrin and GAPDH by their respective siRNA targeting sequences. Right panel: Immunofluorescence staining for αv integrin showing αv integrin knockdown, but not GAPDH knockdown, decreases focal adhesion assembly as pEPCs bind to OPN in culture. The arrows represent punctate immunostaining for the signals representing focal adhesion formation at the base of pEPCs (width of field = 140 μm). B) Adhesion assays were conducted with bovine OPN (bOPN), recombinant rat OPN with an intact RGD sequence (RGD), bovine fibronectin (bFN), and type I collagen (COL I). Adherent cells were fixed, stained with Amido black, and quantified. Bovine serum albumin (BSA) served as a negative control and Type I collagen served as a positive control. Values represent absorbance readings (595 nm; 3×3 wells/data point). C) Immunofluorescence staining of control (non-treated) pEPCs, and pEPCs treated with αv or GAPDH siRNAs (75 nM, 48 h transfection) demonstrate that silencing of αv integrin reduces vinculin and paxillin incorporation into focal adhesions as pEPCs attach to the underlying matrix. A nonrelevant immunoglobulin IgG (width of field = 140 μm).

Article Snippet: After washing with PBS containing 0.3% vol/vol Tween 20, cells were blocked with 10% vol/vol goat serum and incubated overnight at 4°C with 2 μg/μl rabbit polyclonal antibodies to von Willebrand factor (vWF; BD Biosciences, San Jose, CA), platelet endothelial cell adhesion molecule (PECAM-1; Santa Cruz, Dallas, Texas), vascular endothelial cadherin (VE-cadherin; Enzo Life Sciences, Farmingdale, NY), αv integrin subunit (AB1930; Chemicon, Temecula, CA), and mouse antibodies to focal adhesion kinase (FAK; BD Transduction Laboratories, Lexington, KY), vinculin (Sigma-Aldrich), and paxillin (Santa Cruz).

Techniques: Western Blot, Immunofluorescence, Staining, Immunostaining, Recombinant, Sequencing, Negative Control, Positive Control, Transfection

Journal: Reproduction (Cambridge, England)

Article Title: OPN Binds Alpha V Integrin to Promote Endothelial Progenitor Cell Incorporation into Vasculature

doi: 10.1530/REP-19-0358

Figure Lengend Snippet: OPN increases incorporation of pEPCs into sprouting HUVEC networks. A): DiI labeled HUVECs were allowed to invade in the presence of 0 μg/ml (w/o bOPN) or 100 μg/ml bOPN. B): unlabeled HUVECs were seeded along with DiI labeled pEPCs onto collagen gels, and soluble bOPN was added to culture medium in the bottom chamber at the concentrations indicated. Cultures were fixed, stained with DAPI, and the number of invading HUVEC (left panel) and pEPCs (right panel) was quantified. Four fields from each treatment group were used to obtain average number of invading cells per 1 mm2 field (+/− SD). Data shown are representative of 4 independent experiments (bars that are significantly different do not share the same letter). C-D) Alpha v integrin knockdown does not affect HUVEC and/or pEPC invasion in the absence of bOPN. DiI-labeled EPCs expressing no siRNA (Control) or siRNA directed to αv integrin (αv--KD) or GAPDH (GAPDH-KD) were co-cultured on the surface of collagen gels C): Total cell (HUVEC and pEPC) invasion in the absence of bOPN. D): pEPC invasion in the absence of bOPN. Four fields from each treatment group were used to obtain average number of invading cells per 1 mm2 field (+/− SD). Each experiment was performed in triplicate (p=0.10). E-F) bOPN requires αv integrin expressed on pEPCs to increase incorporation of pEPCs into sprouting HUVEC networks. DiI-labeled pEPCs expressing no siRNA (Control) or siRNA directed to αv integrin (αV-KD) or GAPDH (GAPDH-KD) were co-cultured in the presence of 100μg/ml soluble bOPN added to the culture medium in the bottom chamber. E): Total cell (HUVEC and pEPC) invasion in the presence of bOPN. F): pEPC invasion in the presence of bOPN. Four fields from each treatment group per experiment were used to obtain average number of invading cells per 1 mm2 field (+/− SD). Data shown represent 3 independent experiments (P<0.05).

Article Snippet: After washing with PBS containing 0.3% vol/vol Tween 20, cells were blocked with 10% vol/vol goat serum and incubated overnight at 4°C with 2 μg/μl rabbit polyclonal antibodies to von Willebrand factor (vWF; BD Biosciences, San Jose, CA), platelet endothelial cell adhesion molecule (PECAM-1; Santa Cruz, Dallas, Texas), vascular endothelial cadherin (VE-cadherin; Enzo Life Sciences, Farmingdale, NY), αv integrin subunit (AB1930; Chemicon, Temecula, CA), and mouse antibodies to focal adhesion kinase (FAK; BD Transduction Laboratories, Lexington, KY), vinculin (Sigma-Aldrich), and paxillin (Santa Cruz).

Techniques: Labeling, Staining, Expressing, Cell Culture