fab3869a  (R&D Systems)

Journal: Nature Communications

Article Title: Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1038/s41467-024-49718-8

Figure Lengend Snippet: a Percentage of PD-L1 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 23), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 27). b Percentage of CD112 + cells within the indicated cancer cells ( n = 12 tumors per group). c Percentage of Gal9 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 17), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). d Percentage of CD80 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 24), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 18), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). e Percentage of CD155 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 10), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 9), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 12). f , h Representative immunofluorescence images of Ecad + (green), f CD80 + or h CD155 + (red), and DAPI nuclear (blue) staining in the indicated patient cSCCs. Scale bar, 100 µm. g Percentage of CD80 + cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). i Percentage of CD155 + cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Each dot indicates the average quantification of at least five fields from different tumor regions. j Percentage of CD80 − Ecad + , CD80 + Ecad + , and CD80 + Ecad − cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). k Percentage of CD155 − Ecad + , CD155 + Ecad + , and CD155 + Ecad − cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Data are represented as the mean ± SD ( a – e ) or ± SEM ( g , i , j , k ), and n values indicate independent tumors ( a – e , g , i ). P values are determined by one-way ANOVA with Dunnett’s ( a – e ) or Tukey’s ( g , i ) multiple comparison tests. See Supplementary Fig. for the gating strategy ( a – e ). Source data are provided as a Source Data file.

Article Snippet: For cell-surface staining, cells were blocked with 1 mg/ml IgG (Sigma, I5381) and stained with a cocktail of cell-surface antibodies in staining buffer (5% FBS in PBS) for 30 min at 4 °C: from Biolegend, CD11b-APC 1:250 (M1/70, 101211), CD11b-PE/Cy7 1:250 (M1/70, 101215), CD152 (CTLA-4)-PE/Cy7 1:250 (UC10-4B9, 106313), CD155-PE/Cy7 1:200 (TX56, 131511), CD223 (LAG-3)-PE/Cy7 1:250 (C9B7W, 125225), CD226 (DNAM-1)-PE/Cy7 1:250 (10E5, 128811), CD25-PE/Cy7 1:200 (PC61, 102015), CD274 (PD-L1)-PE/Cy7 1:200 (10F.9G2, 124313), CD279 (PD-1)-APC/Cy7 1:250 (29F.1A12, 135223), CD28-PE/Cy7 1:250 (37.51, 102125), CD3ε-APC 1:200 (145-2C11, 100311), CD366 (TIM-3)-PE/Cy7 1:250 (B8.2C12, 134009), CD4-PE/Cy7 1:200 (RM4-5, 100528), CD49f (α6-integrin)-FITC 1:10 (GoH3, 313605), CD69-PE/Cy7 1:200 (H1.2F3, 104511), CD8a-PE 1:200 (53-6.7, 100707), CD80-PE/Cy7 1:250 (16-10A1, 104733), F4/80-APC/Cy7 1:200 (BM8, 123118), Galectin9-PE/Cy7 1:250 (108A2, 137913), Ly-6G/Ly-6C (Gr-1)-PE/Cy7 1:250 (RB6-8C5, 108415), Ly-6C-PE/Cy7 1:250 (HK1.4, 128017), Ly-6G-APC 1:250 (1A8, 127613), NK-1.1-PE 1:200 (PK136, 108707), TIGIT (Vstm3)-PE/Cy7 1:250 (1G9, 142107); from BD Bioscience, CD11b-PE 1:250 (M1/70, 557397); from eBioscience, CD206-APC 1:200 (MR6F3, 17-2061-80), CD326 (EpCAM)-APC-eF780 1:400 (G8.8, 47-5791-82); from TONBO, CD45-PE 1:350 (30-F11, 50-0451); from R&D Systems, CD112 (Nectin-2)-APC 1:200 (829038, FAB3869A).

Techniques: Immunofluorescence, Staining, Comparison

Journal: Scientific Reports

Article Title: Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

doi: 10.1038/s41598-017-10403-0

Figure Lengend Snippet: PVR and Nectin2 are mainly found as intracellular pool in MM cells. ( a ) CD38 + CD138 + malignant PCs derived from BM aspirates of MM patients (n = 34) were analysed for PVR and Nectin2 surface and total (surface plus intracellular) expression before and after fixation and permeabilization, respectively. Cells were acquired using FACSCanto flow cytometer (BD Biosciences). Each dot represents a single patient, ****p < 0.001, Wilcoxon matched pairs test. ( b ) PVR (left panel) and Nectin2 (right panel) surface and total (surface plus intracellular) expression was analysed on MM cell lines before and after fixation and permeabilization, respectively. Cells were acquired using FACSCalibur flow cytometer (BD Biosciences). Data represent the means ± SD of PVR and Nectin2 from three independent experiments. *p < 0.05, Student T test. MFI: mean fluorescence intensity.

Article Snippet: Surface ligands expression on patient-derived PCs was evaluated by means of PE-conjugated anti-PVR mAb (Biolegend, SKII.4), and APC-conjugated anti-Nectin2 mAb (R&D Systems, FAB2229A) after gating on the CD38 + CD138 + PC population.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

doi: 10.1038/s41598-017-10403-0

Figure Lengend Snippet: SUMOylation controls PVR but not Nectin2 surface expression in MM cell lines. ( a–c ) Inhibition of the SUMO pathway was achieved by means of overnight treatment with 25μg/mL Ginkgolic Acid (GA). The efficacy of treatment was verified by means of western blot analysis on total cell lysates ( a ). ( b , c ) PVR and Nectin2 surface expression was evaluated on ARK and OPM2 cell lines by immunofluorescence and FACS analysis using FACSCalibur flow cytometer (BD Biosciences). One out of three independent experiments ( b ) and means ± SD of PVR and Nectin2 MFI from three independent experiments ( c ) are shown. **p < 0.01, ***p < 0.001, Two-way ANOVA. ( d–f ) Inhibition of the SUMO pathway was achieved by means of UBC9 gene silencing. Silencing efficiency was verified by means of western blot analysis on total cell lysates ( d ). ( e , f ) PVR and Nectin2 surface expression was evaluated on ARK and OPM2 cell lines by immunofluorescence and FACS analysis using FACSCanto flow cytometer (BD Biosciences). One out of three independent experiments ( e ) and means ± SD of PVR and Nectin2 MFI from three independent experiments ( f ) are shown. **p < 0.01, ***p < 0.001, Two-way ANOVA.

Article Snippet: Surface ligands expression on patient-derived PCs was evaluated by means of PE-conjugated anti-PVR mAb (Biolegend, SKII.4), and APC-conjugated anti-Nectin2 mAb (R&D Systems, FAB2229A) after gating on the CD38 + CD138 + PC population.

Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Flow Cytometry

Journal: Scientific Reports

Article Title: Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

doi: 10.1038/s41598-017-10403-0

Figure Lengend Snippet: Ginkgolic Acid treatment up-regulates PVR but not Nectin2 surface expression in malignant PCs. Malignant PCs were treated overnight with 25 μg/mL of GA or vehicle alone (DMSO), and PVR ( a ) and Nectin2 ( b ) surface expression was evaluated on cells gated as in Supplementary Fig. . Data from two representative patients are shown in left panels. Data from 9 patients analysed are shown in right panels. Each dot represents a single patient. ***p < 0.001 Wilcoxon matched pairs test.

Article Snippet: Surface ligands expression on patient-derived PCs was evaluated by means of PE-conjugated anti-PVR mAb (Biolegend, SKII.4), and APC-conjugated anti-Nectin2 mAb (R&D Systems, FAB2229A) after gating on the CD38 + CD138 + PC population.

Techniques: Expressing