Journal: Nature Communications

Article Title: The molecular and functional landscape of resistance to immune checkpoint blockade in melanoma

doi: 10.1038/s41467-023-36979-y

Figure Lengend Snippet: A ssGSEA score for Reactome_IFNγ_Signaling in BSA control and IFNγ-treated PD1 PROG cell lines with ( n = 6; orange) and without ( n = 15; blue, not including SCC16-0016) intrinsic IFNγ activity. The dotted line aligns with the lowest ssGSEA score in the IFNγ-treated PD1 PROGs, and solid lines indicate the median ssGSEA scores. B Subset of top-scoring gene sets (GSEA Pre-Ranked; Hallmark gene set collection and melanoma-specific transcriptome signatures) in PD1 PROGs with ( n = 6) compared to without ( n = 15) intrinsic IFNγ activity (Supplementary Data ). C Hierarchical clustering of PD1 PROG cell lines ( n = 22) with Euclidean distance based on protein expression of Melan-A, MITF, SOX10, and AXL. The transcriptome melanoma clusters defined according to ref. are also shown. PD1 PROG cell lines with intrinsic IFNγ signaling ( n = 6) are highlighted in red. D Plots showing IRF1 protein expression (derived from the densitometric normalized protein data after log 2 transformation and z score calculation) in PD1 PROGs with ( n = 6) and without ( n = 5) intrinsic IFNγ activity. The relative cell surface expression (median fluorescence intensity stained divided by fluorescence minus one control, MFI/FMO) of PD-L1, PD-L2, and MHC-I in PD1 PROGs with ( n = 6) and without ( n = 16) intrinsic IFNγ activity. Data compared using two-sided Mann–Whitney test, p -values shown. E Differentially expressed secreted cytokines (FDR-adjusted p -value < 0.1, dotted line) in control (BSA)-treated PD1 PROGs with (IFNγ-high; n = 6) or without (IFNγ-low; n = 15) intrinsic IFNγ signaling are highlighted in red. The comparison of secreted cytokine expression was performed using log 2 transformed fluorescence intensity values (Supplementary Data ). F Differentially expressed secreted cytokines (FDR-adjusted p -value < 0.1) in control (BSA)-treated PD1 PROGs versus IFNγ-treated PD1 PROG cell lines ( n = 21; 1000 U IFNγ/ml for 24) are highlighted in red. The comparison of cytokine expression was performed using log 2 transformed fluorescence intensity values (Supplementary Data ). Source data are provided as a Source Data file.

Article Snippet: Western blots were probed with antibodies against ß2-microglobulin (1:1000; D8P1H; Cell Signaling Technology; Cat No. 12851), CIITA (1:1000; Cell Signaling Technology; Cat No. 3793), STAT1 (1:1000; 9H2; Cell Signaling Technology; Cat No. 9176), phospho-STAT1 Ser727 (1:1000; D3B7; Cell Signaling Technology; Cat No. 8826), JAK1 (1:1000, Cell Signaling Technology; Cat No. 3332), JAK2 (1:1000, E4Y4D; Cell Signaling Technology; Cat No. 74987), IRF1 (1:1000; D5E4; Cell Signaling Technology; Cat No. 8478), AXL (1:200; R&D systems; Cat No. AF154), MLANA/Mart-1 (1:1000; Cell Signaling Technology; Cat No. 34511), MITF (1:1000; C5; Calbiochem; Cat No. OP126L), SOX10 (1:1000; D5V9L; Cell Signaling Technology; Cat No. 89356), and ß-Actin (1:6000; AC-74; Sigma-Aldrich; Cat No. A5316).

Techniques: Control, Activity Assay, Expressing, Derivative Assay, Transformation Assay, Fluorescence, Staining, MANN-WHITNEY, Comparison

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 1. Specificity of a-ADAM10 monoclonal antibodies. (A) Alignment of mouse, human and bovine ADAM10 cysteine-rich domain sequences (AA 551– 646). In the human and bovine sequences, only residues not homologous to mouse are shown. (B) Comparison of binding of mouse hybridoma (fusion) and isolated cell clone supernatants to serially diluted, immobilised bovADAM10 ECD by ELISA. Binding of non-immunised mouse serum (control) is shown for comparison. (C) Binding of endogenous huADAM10 by a-ADAM10 hybridoma clones, or the R&D ADAM10 mAb 1427, was compared by immunoprecipitation from equivalent HEK293 cell lysates and western blotting with an a-ADAM10 pAb; u, unprocessed; p, processed ADAM10. (D) The specificity of 8C7 for ADAM10 was tested by immunoprecipitation from lysates of ADAM10 knockout (2/2) and Wt (+/+) mouse embryonic fibroblasts (MEFs), and a-ADAM10 pAb western blot.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Bioprocessing, Comparison, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay, Control, Clone Assay, Immunoprecipitation, Western Blot, Knock-Out

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 2. Co-staining of cells with ADAM10 mAb 8C7 and ephrin-A5-Fc reveals colocalisation and co-internalisation with EphA3. (A) EphA3/ HEK293 cells were incubated on ice with Alexa647–8C7 mAb and fixed for imaging (0 min) or first allowed to warm to 37˚C for 60 min. (B) Cells were labelled with Alexa647–8C7 and with Alexa488–ephrin-A5-Fc and fixed immediately (0 min) or incubated at 37˚C with a-humanFc to cluster ephrin- A5-Fc for the indicated time periods before fixation. The insets are enlarged images of the regions within the dotted lines. Cells incubated for 60 min with Alexa488–ephrin-A5-Fc alone are shown as a control in the bottom panels. Scale bars: 25 mm.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Staining, Incubation, Imaging, Control

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 3. Site-directed mutagenesis of the ADAM10 substrate-binding pocket disrupts mAb binding. (A) Structure of the bovine ADAM10 D and C domains showing the location of key residues targeted by site-directed mutagenesis. (B) Comparison of aADAM10 mAb binding to Wt and substrate-binding pocket mutant huADAM10. Alanine substitutions at Glu 573, 578 and 579 (3EA) or at residues 617 and 618 (617AA) were made in huADAM10-GFP, and Wt and mutant constructs were transfected into ADAM102/2 MEFs (control: untransfected). Binding of a-ADAM10 mAbs was assessed by immunoprecipitation from equivalent cell lysates, and western blotting with a-ADAM10 pAb (non-relevant lanes removed; the altered molecular mass pattern reflects the GFP-tagged huADAM10). The graph shows binding of 8C7 and 3A8 relative to the R&D mAb, determined by densitometry (one-way ANOVA; **P,0.01 compared to R&D sample; n.s., not significant; n53).

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Mutagenesis, Binding Assay, Comparison, Construct, Transfection, Control, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 5. ADAM10 mAb 8C7 inhibits EphA3 phosphorylation in response to stimulation by cell-bound ephrin. (A) 293/EphA3 cells were pretreated with 0, 10 and 100 mg/ml of 8C7 mAb for 2 h and stimulated for the indicated times. a-EphA3 immunoprecipitates from the cell lysates were analysed by western blot with a-phosphotyrosine (pY) and a-EphA3 antibodies as indicated. A representative image from four experiments is shown. (B) EphA3 phosphorylation relative to EphA3 protein levels was calculated from replicate experiments as described in A, using densitometry analysis. Graph shows means 6 s.e.m., n54. (C) 8C7 does not inhibit EphA3 phosphorylation induced by soluble clustered ephrin-A5. EphA3/293 cells, pre-incubated with or without 8C7 (100 mg/ml) for 2 hours, were stimulated for 20 min with pre-clustered ephrin-A5-Fc, or left unstimulated, as indicated. EphA3 immunoprecipitates from cell lysates were analysed by western blotting as in A.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Phospho-proteomics, Western Blot, Incubation

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 6. ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell repulsion. (A) EphB2/HEK293 cells labelled with Cell Tracker Green were pre-treated with vehicle (Cont), 8C7 (50, 200 or 400 mg/ml), or with GM6001 (GM, 50 mM), and plated onto coverslips pre-coated with fibronectin and stripes of alexa594-labelled ephrin-A5-Fc. As a comparison, cells expressing a signalling-deficient EphB2 mutant (DICD) were also used. After 18 hours the cells were imaged by fluorescence microscopy, from which examples are shown (8C7, 400 mg/ml). Scale bar: 250 mm. (B) The percentage of cells adhering to ephrin stripes was calculated from ,20 images for each treatment; the graph shows the averages 6 s.e.m. from three experiments. (C) 8C7 inhibits ephrin-A5-induced EphB2 phosphorylation. Effects of 8C7 treatment on activation of EphB2/HEK293 cells by ephrin-A5/HEK293 cells was assessed as in Fig. 5A, following stimulating for 40 minutes.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Comparison, Expressing, Mutagenesis, Fluorescence, Microscopy, Phospho-proteomics, Activation Assay