Journal: International Journal of Molecular Sciences

Article Title: ADAM12-Generated Basigin Ectodomain Binds β1 Integrin and Enhances the Expression of Cancer-Related Extracellular Matrix Proteins

doi: 10.3390/ijms25115871

Figure Lengend Snippet: rsBSG binds β1 integrin and enhances integrin-dependent cell migration and gelatin degradation. ( A , B ) Co-immunoprecipitation of rsBSG and β1 integrin in HT1080 cells treated with rsBSG for 2 h before lysis. β1 integrin was immunoprecipitated using AIIB2 β1 integrin antibody and co-immunoprecipitated rsBSG was detected by western blot using an antibody against the strep-tag ( A ), whereas rsBSG was immunoprecipitated using the strep-tag antibody and co-immunoprecipitated β1 integrin was detected using the rat monoclonal AIIB2 anti-β1-integrin antibody ( B ). Total cell lysate (TCL) was tested for β1 integrin expression and the presence of rsBSG protein in the same blots. Control rat IgG immunoprecipitation served as negative control in both settings. ( C ) HT1080 cells were starved over night before being stimulated with 10 ug/mL BSA or rsBSG for 30 min. Samples were analyzed by western blot against p397FAK or total FAK. Actin served as an internal loading control. ( D ) Quantification of p397FAK relative to total FAK. ( E ) HeLa cells were starved overnight before being stimulated with 10 ug/mL BSA or rsBSG for 60 min. Cells were stained with anti-pTyr antibody (4G10), scale bar = 10 µm. ( F ) In situ gelatinase assay of HT1080 cells grown on Oregon green-labeled gelatin and treated with either BSA or rsBSG with either the AIIB2 antibody blocking β1 integrin activity or the metalloprotease inhibitor GM6001 for 6 h. Black areas represent gelatin degradation; scale bar = 50 µm. ( G ) Quantification of the area of gelatin degradation shown in ( F ), measured from 20 images using MetaMorph software. ( H ) In situ gelatinase assay performed as in F; cells were treated with 10 ug/mL BSA or BSG D1. ( I ) Quantification of gelatin degradation assay from ( H ), quantified as in ( F ). For all graphs, values represent means ± SEM from at least three independent experiments. * p < 0.05; *** p < 0.005, Student’s t -test or ANOVA.

Article Snippet: Mouse monoclonal anti-Strep tag (SAB2702216) and rat monoclonal anti-β1 integrin (AIIBII) antibodies were from Sigma-Aldrich (St. Louis, MO, USA), mouse monoclonal antibody against actin (MAB1501) were from Millipore Chemicon (Burlington, MA, USA), rabbit anti-p397 FAK (44-625G) from Invitrogen (Waltham, MA, USA), mouse anti-phospho Tyrosine (clone 4G10) (16-452) and Rabbit anti-fibronectin (MAB2033) from Millipore (16-452) (Burlington, MA, USA), mouse anti-FAK (610088) from BD Bioscience (Franklin Lakes, NJ, USA).

Techniques: Migration, Immunoprecipitation, Lysis, Western Blot, Strep-tag, Expressing, Negative Control, Staining, In Situ, Labeling, Blocking Assay, Activity Assay, Software, Degradation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5β1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling

doi: 10.1186/s13046-023-02930-8

Figure Lengend Snippet: Effects of MS-275 on β1 Integrin expression and activity ( A-B ) Adhesion assays of mesenchymal-like MeT5A pretreated with anti-Integrin α5 and -Integrin α4 blocking antibodies. Results are shown as relative number of adhered EOCs (GFP-SKOV3 cells top, GFP-OVCAR-3 cells, bottom) on the MeT5A monolayer. Adherent SKOV3 cells were analyzed in 3 fields/sample. Each experiment was performed at least 3 times in triplicate. Mesenchymal-like MeT5A cells treated with MS-275 (250 nM) for 72 hours. C RT-qPCR showing the expression of β1 and α5 Integrin subunits from total RNA of mesenchymal-like MeT5A cells treated with MS-275 (250 nM) for 72 h. Bars represent means±SEM of 5 independent experiments. D Immunofluorescence showing mesenchymal-like MeT5A cells treated with MS-275 (250 nM) for 72 hours. Fixed cells were stained with an antibody against total β1 Integrins or against active β1 Integrins (9EG7). The quantification of the experiment is shown on the right. Mander’s colocalization M2 coefficients were measured using the JACoP plugin on ImageJ. At least 10 images were quantified per experiment. Confocal images are shown from one representative experiment of three performed. Scale bar: 20 μm, E , F left, flow cytometry experiments showing the plasma membrane expression total β1 Integrin ( E ) and of active β1-Integrin detected using the monoclonal antibody HUTS21 ( F ). The fluorescence intensity profiles measured through flow cytometry depict a representative experiment. Active β1-Integrin in untreated MeT5A cells appears in blue, whereas in MS-275 treated cells (250 nM) it appears in red. Light-grey profiles depict negative controls. Right, histograms show mean fluorescence intensities (MFI) of β1 Integrin ( E ) and active β1 Integrin stainings ( F ). Bars represent means ± SEM of 5 experiments. Differences were considered significant at P < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001)

Article Snippet: Antibodies for Immunofluorescence experiments were rabbit anti-collagen (NB600–480) from Novus Biological, (Litterton, CO, USA); rabbit anti- FN (ab2413), mouse anti- Integrin β1 (ab30394) from Abcam, (Cambridge, UK) mouse anti-integrin β1(clone HUTS21), rat anti-Integrin β1 (clone 9EG7, 550,531) BD Pharmigen (Franklin Lakes, NJ, USA), Cy3-conjugated anti-rat secondary antibodies (Jackson ImmunoResearch, 112–165-003), anti-rabbit Alexa Fluor 488-conjugated (A21206), anti-mouse Alexa Fluor 488-conjugated (A32723), Alexa fluor Phalloidin 647 (A2228), Rhodamine Phalloidin (R415), Hoechst 33342 (H21492) from Thermo Fisher Scientific.

Techniques: Expressing, Activity Assay, Blocking Assay, Quantitative RT-PCR, Immunofluorescence, Staining, Flow Cytometry, Membrane, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5β1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling

doi: 10.1186/s13046-023-02930-8

Figure Lengend Snippet: MS-275 hampers actin cytoskeletal organization and impacts the MeT5A/SKOV3 cell adhesion by downregulating Talin-1 expression. A Representative Western blot experiment showing expression of Talin-1 from cell lysates of MeT5A cells treated MS-275 (250 nM) for 72 hours. HSP90 was used as a loading control. One of three experiments is shown. Quantification of the experiments is shown below. B Immunofluorescence of primary mesenchymal-like MCs treated with MS-275 (250 nM) for 72 hours showing the expression of Actin filaments stained with phalloidin (red). Nuclei are shown in blue (DAPI). Scale bar: 10 μm. C Western blot showing Talin-1 expression in Talin-1 silenced MeT5A cells. HSP90 was used as a loading control. One of three experiments is shown. Quantification of the experiments is shown on the right. D Immunofluorescence showing mesenchymal-like MeT5A cells stained with an antibody against active β1 Integrins (9EG7) (top) or against β1 Integrins (bottom). Mander’s colocalization M2 coefficients were measured using the JACoP plugin on ImageJ. At least 10 images were quantified per experiment. The quantification of the experiment is shown at the bottom. Confocal images are shown from one representative experiment of three performed. Scale bar: 20 μm ( E ) Immunofluorescence of Talin-1 silenced MeT5A cells stained with phalloidin (grey) and DAPI (blue). Scale bar: 10 μm. Representative images are shown from one of three experiments performed. F FN-1 staining of decellularized matrices of MeT5A cells treated with genetically silenced for Talin-1 for 72 hours. Nuclei are stained with DAPI. Decellularized matrices are shown on the right. Representative images are shown from one of three experiments performed. G Adhesion assays on Talin-1 silenced MeT5A cells. Results are shown as relative number of adherent GFP-SKOV3 cells on Talin-1 silenced MeT5A monolayers. Adherent SKOV3 cells were evaluated in 3 fields/sample. Bars represent the means±SEM of four experiments. Differences were considered significant at P < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001)

Article Snippet: Antibodies for Immunofluorescence experiments were rabbit anti-collagen (NB600–480) from Novus Biological, (Litterton, CO, USA); rabbit anti- FN (ab2413), mouse anti- Integrin β1 (ab30394) from Abcam, (Cambridge, UK) mouse anti-integrin β1(clone HUTS21), rat anti-Integrin β1 (clone 9EG7, 550,531) BD Pharmigen (Franklin Lakes, NJ, USA), Cy3-conjugated anti-rat secondary antibodies (Jackson ImmunoResearch, 112–165-003), anti-rabbit Alexa Fluor 488-conjugated (A21206), anti-mouse Alexa Fluor 488-conjugated (A32723), Alexa fluor Phalloidin 647 (A2228), Rhodamine Phalloidin (R415), Hoechst 33342 (H21492) from Thermo Fisher Scientific.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5β1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling

doi: 10.1186/s13046-023-02930-8

Figure Lengend Snippet: Schematic representation of the effect of HDAC1/2 inhibition on MC/EOC adhesion. HDAC1/2 inhibition downregulates the expression of Talin-1 and other cytoskeletal regulators. This alters the Actin network leading to α5β1 Integrin inactivation and impairing FN-1 secretion and eventually inhibiting MC/EOC adhesion

Article Snippet: Antibodies for Immunofluorescence experiments were rabbit anti-collagen (NB600–480) from Novus Biological, (Litterton, CO, USA); rabbit anti- FN (ab2413), mouse anti- Integrin β1 (ab30394) from Abcam, (Cambridge, UK) mouse anti-integrin β1(clone HUTS21), rat anti-Integrin β1 (clone 9EG7, 550,531) BD Pharmigen (Franklin Lakes, NJ, USA), Cy3-conjugated anti-rat secondary antibodies (Jackson ImmunoResearch, 112–165-003), anti-rabbit Alexa Fluor 488-conjugated (A21206), anti-mouse Alexa Fluor 488-conjugated (A32723), Alexa fluor Phalloidin 647 (A2228), Rhodamine Phalloidin (R415), Hoechst 33342 (H21492) from Thermo Fisher Scientific.

Techniques: Inhibition, Expressing