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 Cell International

Article Title: Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

doi: 10.1186/s12935-024-03417-y

Figure Lengend Snippet: Analysis of αVβ6 expression. ( A ) ITGB6 proteomic expression profile in normal tissue and primary tumor (data from UALCAN) ( n = 71–108; *** p < 0.001; Two sample t test). ( B ) RT-qPCR analysis of ITGB6 mRNA expression; β-actin was used as housekeeping gene. The (αVβ6+) cell line HT-29 was used as reference ( n = 3–8; *** p < 0.001; One-way ANOVA followed by Tukey’s assay). ( C - D ) β6 integrin subunit protein expression assessed by Western-Blot; tubulin was used as a loading control ( n = 3–7; *** p < 0.001; One way ANOVA followed by Tukey’s assay). Data are presented as mean ± SEM

Article Snippet: 40 µg of protein lysate was loaded to 7.5% non-reducing SDS PAGE and after 150 min of migration and 90 min of blotting, PVDF membrane was saturated with 5% (w/v) solution of non-fat powered milk in TBST (Tris buffer solution with 0.1% Tween-20) for 1 h. Membrane was incubated overnight at 4 °C with either 1:100 mouse anti-human β6 integrin subunit antibody (Merck Millipore corp, USA, 407,317) or 1:1000 mouse anti-α-tubulin antibody (Santa Cruz Biotechnology, 23,948), followed by incubation with 1:2000 anti-mouse IgG HRP-conjugated secondary antibody (Cell signaling technology, 7076 S) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cancer Cell International

Article Title: Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

doi: 10.1186/s12935-024-03417-y

Figure Lengend Snippet: Multicellular tumor spheroid characterization. ( A ) Typical cryosection images of H10 and H5M5 spheroids after KI-67 immunohistochemistry staining (scale bar = 150 μm). ( B ) Typical immunofluorescence images of H10 and H5M5 spheroids cryosections stained with antibodies against αVβ6 integrin, cytokeratin 19, E-cadherin, vimentin, and fibronectin at day 4 post-seeding. Proteins of interest are in red and nuclei are in bleu after counterstaining with Vectashield-DAPI (scale bar = 100 μm at x40 and 40 μm at x100). ( C ) Relative quantification of immunofluorescence markers expression using ImageJ software ( n = 3–4; * p < 0.05, ** p < 0.01; Two sample t test). Data are presented as mean ± SEM

Article Snippet: 40 µg of protein lysate was loaded to 7.5% non-reducing SDS PAGE and after 150 min of migration and 90 min of blotting, PVDF membrane was saturated with 5% (w/v) solution of non-fat powered milk in TBST (Tris buffer solution with 0.1% Tween-20) for 1 h. Membrane was incubated overnight at 4 °C with either 1:100 mouse anti-human β6 integrin subunit antibody (Merck Millipore corp, USA, 407,317) or 1:1000 mouse anti-α-tubulin antibody (Santa Cruz Biotechnology, 23,948), followed by incubation with 1:2000 anti-mouse IgG HRP-conjugated secondary antibody (Cell signaling technology, 7076 S) for 1 h at room temperature.

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Expressing, Software

Journal: International journal of molecular sciences

Article Title: Oleuropein Stimulates Migration of Human Trophoblast Cells and Expression of Invasion-Associated Markers.

doi: 10.3390/ijms25010500

Figure Lengend Snippet: Figure 5. Effect of oleuropein (OLE) on the expression of integrin subunits α1 (ITGα1) (A) and β1 (ITGβ1) (B) in HTR-8/SVneo cells at the protein level, using the CELISA method. * p < 0.05.

Article Snippet: After blocking, 50 μL of each primary antibody for α1 integrin subunit (MAB5676, source: mouse, 1:400, R&D Systems, MN, USA) or β1 integrin subunit (AB1952, source: rabbit, 1:500, Millipore Sigma, Burlington, MA, USA) or COX-2 (PA5-27238, source: rabbit, 1:500, Invitrogen, Waltham, MA, USA) was added in PBS with 1% BSA to the wells and incubated overnight at 4 ◦C in a humidified chamber.

Techniques: Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: To pharmacologically block Rho/ROCK pathway and β1-integrin, hPAECs were pretreated with the ROCK inhibitor Y-27632 (#10005583, Cayman Chemical Company, Michigan) and an anti-β1-integrin subunit antibody (#MAB17781, clone P5D2, R&D Systems, Minnesota).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation