Journal: Oncogene
Article Title: Multimerin-2 is a ligand for group 14 family C-type lectins CLEC14A, CD93 and CD248 spanning the endothelial pericyte interface
doi: 10.1038/onc.2017.214
Figure Lengend Snippet: CD248 binds to MMRN2 in a distinct region from CLEC14A and CD93 binding. ( a ) HEK293T transfected with GFP, CLEC14A-GFP, CD248-GFP, and chimeras CLEC14A CD248(sushi) and CLEC14A CD248(CTLD) were lysed and far western blotted with MMRN2 FL and western blotted with anti-GFP. MMRN2 FL binds to CLEC14A and both chimeras CLEC14A CD248(sushi) and CLEC14A CD248(CTLD) but not CD248-GFP or GFP alone. ( b ) Immunoprecipitations of GFP-tagged proteins after cell surface biotinylation. CLEC14A, THBD, CD93 and all four CLEC14A chimeras bind to streptavidin showing they are cell surface expressed. GFP alone was included to demonstrate intracellular proteins are not cell surface biotinylated. ( c ) MMRN2 truncation mutants were transfected into HEK293T and lysates under reducing conditions were far western blotted with mCD248-ECD-Fc, revealing binding to minimal fragment MMRN2 133 –486 . His tag western blot confirmed expression of each MMRN2 protein fragment. ( d ) mCD248-ECD-Fc pull-downs from HUVEC lysates resulted in enrichment of MMRN2 compared with hFc control. ( e ) Immunofluorescence analysis of HUVEC stained with MMRN2 mouse polyclonal antibodies, hFc, mCD248-ECD-Fc or CLEC14A-ECD-Fc. MMRN2 antibody staining partially co-localizes with CD248-ECD-Fc and CLEC14A-ECD-Fc binding; scale bar, 40 μm. ( f ) ELISAs of mCD248-ECD-Fc bound to plate capturing MMRN2 FL His, (** P <0.01 Mann–Whitney test, n =5) error bars represent s.e.m. ( g ) ELISAs of mCD248-ECD-Fc capturing MMRN2 FL His and then binding by CLEC14A-ECD-Fc detected by anti-CLEC14A antibody C2. (** P <0.01 Mann–Whitney test, n =5) error bars, s.e.m. ( h ) Diagram of CLEC14A or CD93 expressed by endothelial cells binding to MMRN2 in the ECM, which in turn is bound by CD248 expressed by fibroblasts or vasculature associated pericytes. CD248, CLEC14A and CD93 binding is due to the CTLD, CD248 binds MMRN2 in the region 133–486, whereas CLEC14A binds in the region 530–624.
Article Snippet: Antibodies: anti-CLEC14A mouse monoclonals C1–C5 generated in our laboratory, anti-CLEC14A sheep polyclonal (R&D Systems, Abingdon, UK, #AF4968), mouse anti-His clone AD1.1 (R&D Systems, #MAB050), anti-GFP mouse clone 3E1 (Cancer Research UK), anti-MMRN2 mouse polyclonal (Abnova/Novus Biologicals, Abingdon, UK, #H00079812-B01P), anti-MMRN2 rabbit polyclonal (Abcam, Cambridge, UK, #ab171314), anti-CD93 mouse clone R139 (Thermo Fisher Scientific, Rugby, UK, #14-0939), anti-CD93 goat polyclonal (R&D Systems, #AF2379), anti-CD248 mouse clone B1.35 (hybridoma supernatants), anti-CD31 mouse clone JC70A (Dako, Cambridge, UK, #M0823), anti-Tubulin mouse clone DM1A (Sigma-Aldrich, Gillingham, UK, #T9026), anti-fibronectin sheep polyclonal (R&D Systems, #AF1918), anti-CD31 rat clone MEC13.3 (BD Bioscience, Oxford, UK, #565629), mouse IgG isotype control (Thermo Fisher Scientific, #10400C), human IgG Fc (Bethyl Laboratories, Cambridge, UK, #P80-104) (sodium azide removed by dialysis in phosphate-buffered saline (PBS)), anti-human IgG Fc HRP conjugated (Sigma-Aldrich, #A0170), anti-mouse HRP (Dako, #P0447), anti-sheep HRP (R&D Systems, #HAF016), anti-goat HRP (Dako, #P0449), streptavidin HRP (GE Healthcare, Amersham, UK, #RPN1231) anti-mouse alexafluor-555 (Thermo Fisher Scientific, #A21425), anti-human IgG alexafluor-555 (Thermo Fisher Scientific, #A21433), anti-mouse alexafluor-488 (Thermo Fisher Scientific, #A11001), anti-rabbit alexafluor-647 (Thermo Fisher Scientific, #A31573), anti-sheep alexafluor-546 (Thermo Fisher Scientific, #A21098), anti-hFc FITC conjugated (Sigma-Aldrich, #F9512), streptavidin R-phycoerthrin (PE) conjugate (Thermo Fisher Scientific, #S-866), fibronectin (Sigma-Aldrich, #F2006).
Techniques: Binding Assay, Transfection, Western Blot, Expressing, Control, Immunofluorescence, Staining, MANN-WHITNEY
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![( A ) H3K27ac and BRG1 ChIP-seq tracks and RNA-seq tracks at PHOX2B and <t>FN1</t> loci in control (red), ARID1A −/− #1 (blue), and ARID1A −/− #2 (black) NGP cells. ( B ) Western blot analysis of ARID1A, an adrenergic maker (PHOX2B), and three mesenchymal markers (FN1, SNAI2, and VIM) in control (Ctrl), ARID1A −/− #1, and ARID1A −/− #2 NGP cells. α-Tubulin was used as a loading control. ( C ) mRNA expression (RPKM of RNA-seq) of core regulatory circuitries of transcription factors specific for adrenergic (ADRN core TFs) or mesenchymal (MES core TFs) cells in control (Ctrl), ARID1A −/− #1, and ARID1A −/− #2 NGP cells. ( D ) GSEA to determine the enrichment of a generic gene signature for mesenchymal tumor in ARID1A mutant NGP cells. Genes are ranked by score and plotted along the x axis as vertical black bars. NES, normalized enrichment score; KO, knockout. ( E ) Cell viability [% relative to N , N ′-dimethylformamide (DMF)–treated cells, y value] of control (red), ARID1A −/− #1 (blue), and ARID1A −/− #2 (black). NGP cells were measured after treatment with various concentrations of cisplatin (0 to 20,000 nM, x value) for 72 hours, and half-maximal inhibitory concentration (IC 50 ) values are indicated. Values are means ± SD of triplicate experiments.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9613/pmc07439613/pmc07439613__aaz3440-F5.jpg)
