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: Journal of Nanobiotechnology

Article Title: Aminolysis-mediated single-step surface functionalization of poly (butyl cyanoacrylate) microbubbles for ultrasound molecular imaging

doi: 10.1186/s12951-024-02806-9

Figure Lengend Snippet: In vitro binding of cRGD-MB to HUVEC under flow conditions. A Fluorescence images of non-stimulated (control) or TNF-α stimulated HUVEC. The nuclei, cell membrane and α v β 3 integrins were stained with DAPI (blue), wheat germ agglutinin-488 (WGA, green) and CD51/61 antibody (red), respectively. Scale bar = 50 μm. B Quantitative analysis indicating significantly higher area fraction of α v β 3 integrin in HUVEC stimulated with TNF-α. Data are represented as mean ± SD. C Schematic showing the in vitro setup of the flow chamber. Petri dishes with cultured HUVEC were connected to a flow chamber, in turn perfused with an MB solution (standard-MB, cRAD-MB, or cRGD-MB) at 0.25 mL/min. D Representative fluorescent images of the three different, rhodamine-labeled MB types binding to TNF-α stimulated HUVEC. The nuclei, cell membrane and MB were labeled with DAPI (blue), WGA (green) and rhodamine (red), respectively. Scale bar: 100 μm. E Number of bound MB per cell. cRGD-MB displayed significantly higher binding to TNF-α stimulated HUVEC compared to both controls. Data represent mean ± SD of three independent MB batches. Statistical comparisons were performed using unpaired t-tests in panel B, while for panel E comparisons were performed using one-way ANOVA. **p < 0.01 and ***p < 0.001

Article Snippet: The tumor sections were fixed with 80% methanol for 5 min at 4 °C followed by the addition of acetone at − 20 °C for 2 min. After fixation, sections were washed 3 times with PBS and incubated overnight with a rabbit anti-α v β 3 integrin antibody (1 μg/ μL; eBioscience, San Diego, California, USA) at a dilution of 1:100 at 4 °C.

Techniques: In Vitro, Binding Assay, Fluorescence, Control, Membrane, Staining, Cell Culture, Labeling

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