mouse anti icam1 bba3  (R&D Systems)

Journal: bioRxiv

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

doi: 10.1101/2024.10.28.620627

Figure Lengend Snippet: A-C Continuous uptake of anti-ALCAM antibody for 15 min at 37°C in the following LB33-MEL cell lines: wild-type (WT, B), stably transfected with empty plasmid (Φ, B), or stably expressing EndoA3-GFP (LB33-MEL EndoA3+, A-C). In (C), cells were transfected with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Quantification of EndoA3 depletion by western blots in Fig EV1A. (A) Airyscan images of Anti-ALCAM (red) and EndoA3-GFP (green). White arrowheads show colocalization between ALCAM and EndoA3. Scale bars: 10 μm (full size image), 1 μm (enlarged cropped areas). (B-C) Quantifications of anti-ALCAM internalization, expressed as fractions of WT condition (B) or siCtrl condition (C). (B) n cells: WT, n = 270; Φ, n = 279; EndoA3+, n = 274. (C) n cells: siCtrl, n = 350; siEndoA3, n = 234. Representative image examples in Fig EV1B-C. D-E Live-cell TIRF images of EndoA3-GFP (stable) and ICAM1-mScarlet (transient) in HeLa (D) and LB33-MEL (E) cells. Time series show enlarged cropped areas extracted from Mov EV1 (D) and Mov EV2 (E). White arrows indicate dynamic co-distribution of both signals. Kymograph in (E) was made along dashed line region in the 2 nd cropped area (D; Mov EV3). Scale bars: 10 μm (full size image), 2 μm (enlarged cropped areas). Data information: All images (A, D and E) are representative of two independent experiments. In (B-C), data are pooled from three independent experiments. Data are presented as median and quartiles. ns, not significant. ****P < 0.0001 (B, Kruskal-Wallis test with Dunn’s multiple comparison test; C, Mann-Whitney test). Source data are available online for this figure.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Negative Control, Western Blot, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

doi: 10.1101/2024.10.28.620627

Figure Lengend Snippet: A Illustration of the SNAP-tag-based BG-labelled antibody uptake assay to study membrane protein endocytosis and retrograde transport. B Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in HeLa cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (HeLa GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. Fluorescence intensity profile was made along the dashed line region in enlarged cropped area, and shows the colocalization of both signals. Scale bar: 20 μm. C-F Retrograde transport of ALCAM and ICAM-1. Continuous BG-labelled anti-ALCAM (C and E) and anti-ICAM1 (D and F) antibody uptake for 4h at 37°C in HeLa GalT-GFP-SNAP cells. (C-D) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Immunodetection made with anti-SNAP, anti-EndoA3 and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex (IB:anti-SNAP) is shown as fractions of siCtrl condition (histogram). Quantification of EndoA3 depletion is shown in Fig EV2E. (E-F) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72h with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection made with anti-SNAP, anti-VPS35, anti-VPS26A and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in Fig EV2F. Data information: In (B), images are from a single experiment. Quantification data (C-F) are pooled from three independent experiments. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (C-D, one sample t test; E-F, RM one-way ANOVA with Dunnett’s multiple comparison test). Source data are available online for this figure.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Membrane, Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Transfection, Negative Control, Immunodetection, Control, Comparison

Journal: bioRxiv

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

doi: 10.1101/2024.10.28.620627

Figure Lengend Snippet: A Live-cell Airyscan images of an IS-like conjugate formed between a CD8 T cell (red) and an adherent stable HLA-A*68012-expressing HeLa cell line transiently expressing ICAM1-EGFP (green). Time series show enlarged cropped areas extracted from Mov EV6. The dashed line contours delineate the contact area between the HeLa cell and the CD8 T cell. White arrowheads show two ICAM1-positive vesicular structures moving towards the contact area. Scale bars: 10 μm (full size image), 2 μm (enlarged cropped areas). B Airyscan images of an IS-like conjugate formed between a CD8 T cell (stained for CD45, yellow) and an adherent stable HLA-A*68012-expressing HeLa cell (stained for microtubules, green) transiently expressing ICAM1-mScarlet (red). White arrowheads indicate recruitment of ICAM1 to the vicinity of CD8 T cell. The fluorescence intensity profile was made along the dashed line region in the enlarged cropped area, and shows enrichment of ICAM1 signal close to the CD8 T cell surface. Scale bars: 20 μm (full size image), 5 μm (enlarged cropped areas). C Airyscan images of an IS-like conjugate formed between a CD8 T cell (stained for CD45, yellow) and a stable HLA-A*68012-expressing HeLa cell (stained for actins, green) transiently expressing ICAM1-mScarlet (red) in suspension. The fluorescence intensity profile was made along the dashed line region. Cell segmentation (white contour) was based on the actin staining for further quantifications (D). Scale bar: 5 μm. D Quantification of relative ICAM1 recruitment to the vicinity of CD8 T cell when an IS-like conjugate is formed between a CD8 T cell and a stable HLA-A*68012-expressing HeLa cell transiently expressing ICAM1-mScarlet in suspension. HLA-A*68012-expressing HeLa cells were transfected for 72h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Western blot analysis of EndoA3 depletion is shown in Fig EV4E. n conjuagates: siCtrl, n = 85; siEndoA3, n = 75. Data information: In (A) and (C), images are representative of three independent experiments. In (B), images are from a single experiment. In (D), data are pooled from three independent experiments. Data are presented as median and quartiles. *P < 0.05 (Kolmogorov-Smirnov test). Source data are available online for this figure.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Expressing, Staining, Fluorescence, Suspension, Transfection, Negative Control, Western Blot

Journal: bioRxiv

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

doi: 10.1101/2024.10.28.620627

Figure Lengend Snippet: Immune synapse components ALCAM and ICAM1 are endocytosed into cancer cells through EndoA3-mediated CIE and are subsequently transported to the TGN in a retromer-dependent retrograde manner. Upon immune synapse formation, immune synapse components, such as ALCAM and ICAM1, are efficiently recruited to the contact area with CD8 T cells, likely from the TGN by polarized re-distribution, which can stabilize the immune synapse and promote CD8 T cell activation (left). Consequently, disruption of EndoA3-mediated CIE or retromer-dependent retrograde transport in cancer cells impair the efficient recruitment of immune synapse components to the contact area with CD8 T cells. In this situation, CD8 T cells expand their contact areas with cancer cells to try to compensate the loss of immune synapse components. The compensation is insufficient, which ultimately results in impaired T cell activation (right).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Activation Assay, Disruption