Journal: bioRxiv

Article Title: Cell adhesion molecule interaction with Piezo1 channels is a mechanism for sub cellular regulation of mechanical sensitivity

doi: 10.1101/602532

Figure Lengend Snippet: COS-7 cells expressing the indicated constructs were studied by FRET/FLIM. a ) Intensity images (left, black and white) and lifetime map (right, rainbow coloured) of Piezo1-mTurquoise2 alone. The plot on the far right is the lifetime distribution. The impact of co-expression of CD31-SYFP2 ( b ), VE-cadherin-mVenus ( c ) or VEGFR2-SYFP2 ( d ) on the intensity and lifetime distribution of Piezo1-mTurquoise2. In a - d the scale bars are 50 μm and the range represented by the rainbow colour bar is 3.5 to 4.2 ns. e - g ) Mean ± s.e.mean lifetimes calculated for n=3 (VE-cadherin and VEGFR2) and n=4 (CD31) independent repeats. Changes in mean ± s.e.mean lifetime over the whole image are plotted for ( e ) Piezo1/CD31 (P = 3.16×10 −5 ), ( f ) Piezo1/VE-cadherin (P = 7.68×10 −4 ) and ( g ) Piezo1/VEGFR2 (P>0.05).

Article Snippet: Proteins were transferred to PVDF membranes and labelled overnight with anti-HA (0.01 μg/mL, Roche clone 3F10), anti-VE-Cadherin (0.5 μg.ml −1 , R&D Systems; MAB9381), anti-VEGFR2 (0.2 μg.ml −1 , R&D Systems; AF357), anti-CD31 (1:1000, Dako; clone JC70A) or anti-β-actin (200 ng.ml −1 , Santa Cruz).

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: Cell adhesion molecule interaction with Piezo1 channels is a mechanism for sub cellular regulation of mechanical sensitivity

doi: 10.1101/602532

Figure Lengend Snippet: COS-7 cells were studied by FRET/FLIM after expressing Piezo1-mTurquoise2 ( a ) with or without CD31-SYFP2 ( b - d ), VE-cadherin-mVenus ( e - g ) or VEGFR2-SYFP2 ( h - j ). Prior to imaging, cells were preconditioned by 24-hr exposure to laminar fluid flow (shear stress = 10 dyn.cm −2 ) followed by stopped flow for 30 min (static) and then a 10-min pulse of repeated flow or no flow. a ) Intensity (left) and lifetime (right) images of Piezo1-mTurquoise2 under 10 min flow conditions. b ) Intensity images of Piezo1-mTurquoise2 (left) and CD31-SYFP2 (middle) and lifetime image of Piezo1-mTurquoise2 (right). Plots show the mean ± s.e.mean lifetimes of Piezo1-mTurquoise2/CD31-SYFP2 under 10 min flow or no flow at c ) intracellular regions (P=0.02, 10 min flow) or d ) cell-cell junctions (P=0.02, 10 min flow; P=0.01 no flow). e ) Intensity images of Piezo1-mTurquoise2 (left) and VE-cadherin-mVenus (middle) and lifetime image of Piezo1-mTurquoise2 (right). The mean ± s.e.mean lifetimes of Piezo1-mTurquoise2/VE-cadherin-mVenus under 10 min flow or no flow at f ) intracellular regions (P>0.05) or g ) cell-cell junctions (P=0.002, 10 min flow; P=0.03, no flow). h ) Intensity images of Piezo1-mTurquoise2 (left) and VEGFR2-SYFP2 (middle) and lifetime image of Piezo1-mTurquoise2 (right). The mean ± s.e.mean lifetimes of Piezo1-mTurquoise2/VEGFR2-SYFP2 under 10 min flow or no flow at i ) intracellular regions (P>0.05) or j ) cell-cell junctions. Mean ± s.e.mean lifetimes were calculated for n=3 independent repeats in all cases. In a , b , e and h the scale bars are 50 μm and the range represented by the rainbow colour bar is 3.5 to 4.2 ns

Article Snippet: Proteins were transferred to PVDF membranes and labelled overnight with anti-HA (0.01 μg/mL, Roche clone 3F10), anti-VE-Cadherin (0.5 μg.ml −1 , R&D Systems; MAB9381), anti-VEGFR2 (0.2 μg.ml −1 , R&D Systems; AF357), anti-CD31 (1:1000, Dako; clone JC70A) or anti-β-actin (200 ng.ml −1 , Santa Cruz).

Techniques: Expressing, Imaging, Shear

Journal: British Journal of Cancer

Article Title: CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells

doi: 10.1038/sj.bjc.6606056

Figure Lengend Snippet: Expression of CXCR3 on human melanoma cells. ( A ) Human melanoma cells isolated from melanoma metastases were stained for surface CXCR3 and analysed by flow cytometry. Grey and bold histogram, CXCR3 staining; black or spotted histogram, isotype staining. ( B ) Melanoma-7 were immunostained with anti-CXCR3 antibody, Alexa488-conjugated anti-mouse as secondary reagent, and for actin with phalloidin-568 and viewed by confocal laser scanning microscopy; bar represents 50 μ m. ( C ) Melanoma-15 was co-cultured with HUVECs and stained with anti-CXCR3 (Alexa-488) and anti-CD144 (Alexa-633) antibodies; bar represents 50 μ m. ( D ) Immunolocalisation of CXCR3 in human skin and melanoma metastasis. Cryosections of human skin and melanoma metastasis were stained with anti-CXCR3 antibody (Alexa-488), anti-CD144 antibody (Alexa-633) and phalloidin-568.

Article Snippet: FITC-conjugated anti-CXCR3 mAb and biotinylated goat IgG anti-human CD144 were purchased from R&D Systems, PE-conjugated anti-CD34 from BD Biosciences (San Jose, CA, USA) and PC5-conjugated anti-CD144 from Immunotech (Marseille, France).

Techniques: Expressing, Isolation, Staining, Flow Cytometry, Confocal Laser Scanning Microscopy, Cell Culture

Journal: British Journal of Cancer

Article Title: CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells

doi: 10.1038/sj.bjc.6606056

Figure Lengend Snippet: Expression of CXCR3 ligands, CXCL9 and CXCL10, in human tumour endothelial cells. ( A ) Immunolocalisation of CXCL9 in human skin and melanoma metastases. Cryosections of normal human skin and melanoma metastasis (lymph node) were stained with anti-CXCL9 antibody (Alexa-488), anti-CD144 antibody (Alexa-568), and phalloidin-568 and viewed by confocal laser scanning microscopy; bar represents 50 μ m. Insert indicates tumour vessel in a higher magnification (dotted structure). ( B , C ) Quantitative Real-time PCR of CXCL9 and CXCL10. Blood ECs, LECs and TuECs were isolated from human skin and melanoma metastasis by cell sorting using anti-CD34, anti-CD144 and anti-podoplanin antibodies, total RNA was isolated, reverse transcribed and corresponding cDNA subjected to TaqMan PCR using commercial probes and primers for CXCL9 and CXCL10. All data shown are mean values and standard error of means from two independent PCR measurements, and have been normalised to the internal control gene B2M (N, normal skin; T, malignant melanoma).

Article Snippet: FITC-conjugated anti-CXCR3 mAb and biotinylated goat IgG anti-human CD144 were purchased from R&D Systems, PE-conjugated anti-CD34 from BD Biosciences (San Jose, CA, USA) and PC5-conjugated anti-CD144 from Immunotech (Marseille, France).

Techniques: Expressing, Staining, Confocal Laser Scanning Microscopy, Real-time Polymerase Chain Reaction, Isolation, FACS, Reverse Transcription, Control