mouse anti apmap  (Novus Biologicals)

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Images of pHrodo-labelled GFP+ Ramos cells of indicated genotypes after 12 h incubation with J774 macrophages with or without anti-CD20. Scale bars, 50 μm. Representative of three biologically independent experiments. b, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes by J774 macrophages with indicated concentrations of anti-CD20, normalized to control (SafeKO) cells in absence of anti-CD20 (n = 4 replicate wells). c, Volcano plot of genome-wide CRISPR knockout screen in Karpas299-Cas9 cells for resistance to treatment with anti-CD47 and macrophages. Dotted line indicates 5% FDR. d, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes following incubation with J774 macrophages and anti-CD47 antibodies or mouse isotype control (mIgG1) antibodies, normalized to control (SafeKO) cells with isotype control antibody (n = 3 cell culture wells). e, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells expressing indicated sgRNAs, incubated for 2 h with J774 macrophages and anti-CD20, anti-CD47, or human (hIgG1) or mouse (mIgG1) isotype control antibodies, normalized to signal in control (SafeKO) cells in anti-CD20 or mIgG1-isotype condition (n = 3 cell culture wells). f, Top, schematic of APMAP structure. Bottom left, APMAP homology model (residues 61–407) (Methods). Bottom right, magnified view of catalytic site of APMAP homology model, showing predicted positioning of residue E103 near the catalytic calcium ion. TMD, transmembrane domain. g, Phagocytosis assay for uptake of pHrodo-labelled indicated Ramos-Cas9 cells by J774 macrophages in the presence of anti-CD20 antibodies, normalized to control (SafeKO) cells (n = 3 cell culture wells). In b, d, e, g, data are mean ± s.d. One-way ANOVA (g) or two-way ANOVA (b, d, e) with Bonferroni correction.

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: Incubation, Phagocytosis Assay, Genome Wide, CRISPR, Knock-Out, Cell Culture, Expressing

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Localization of APMAP-FLAG and APMAPE103A-FLAG to the endoplasmic reticulum in HeLa cells. Scale bar, 20 μm. Calnexin is used as a marker of the endoplasmic reticulum. FLAG staining was representative of two independent experiments. b, Immunoblotting of cell extracts derived from Ramos cells of indicated genotypes expressing indicated APMAP-FLAG constructs. GAPDH served as loading control. Experiment was performed twice. c, d, Phagocytosis assay for uptake of pHrodo-labelled Ramos-Cas9 cells with indicated genotypes by J774 macrophages in the presence of anti-CD20 antibodies. APMAP-F, APMAP-FLAG. TFRCRR, mutant allele of TFRC that localizes primarily to the endoplasmic reticulum47. Phagocytosis index normalized to control (SafeKO) cells. Data represent mean +/− s.d. (n = 4 cell culture wells). One-way ANOVA with Bonferroni correction. e, Immunoblotting of cell extracts that were treated, where indicated, with PNGase F to remove N-glycosylation. Actin served as loading control. Experiment was performed once. f, Phagocytosis assay for uptake of pHrodo-labelled Ramos Cas9 cells expressing indicated sgRNAs and indicated addback constructs by J774 macrophages in the presence of anti-CD20 antibodies. Normalized phagocytosis index was calculated as average total pHrodo Red signal at 5 h for each well, normalized to signal in SafeKO cells at 5 h timepoint. Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction. For gel source data, see Supplementary Figure 1.

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: Marker, Staining, Western Blot, Derivative Assay, Expressing, Construct, Phagocytosis Assay, Mutagenesis, Cell Culture

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Schematic and volcano plot of CRISPR screen in Ramos Cas9 cells for sensitivity to macrophage phagocytosis in the presence of anti-CD20 in cells expressing an sgRNA targeting a Safe locus. Dotted line indicates 5% FDR. A transmembrane gene-enriched sublibrary containing 3,124 genes was used. b, Schematic and volcano plot of CRISPR screen in Ramos Cas9 cells for sensitivity to macrophage phagocytosis in the presence of anti-CD20 in cells expressing an sgRNA targeting the CD47 locus. Dotted line indicates 5% FDR. A transmembrane gene-enriched sublibrary containing 3,124 genes was used. c, Schematic and volcano plot of CRISPRko screen in Ramos Cas9 cells for sensitivity to macrophage phagocytosis in the presence of anti-CD20 and anti-CD47 in cells expressing an sgRNA targeting a Safe locus. Dotted line indicates 5% FDR. A transmembrane gene-enriched sublibrary containing 3,124 genes was used. d, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes by human primary peripheral blood-derived macrophages, from two independent healthy de-identified human donors, in the presence or absence of anti-CD47 antibodies. Phagocytosis index normalized to control (SafeKO) cells without anti-CD47. Data represent mean +/− s.d. (n = 4 cell culture wells). One-way ANOVA with Bonferroni correction. e, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes by J774 macrophages in the presence of anti-CD20 or anti-CD47 antibodies. Where indicated, J774 macrophages were pre-incubated with Fc-blocking antibodies for 45 min on ice. Phagocytosis index normalized to control (SafeKO) cells without antibody analysed in parallel (condition not shown). Data represent mean +/− s.d. (n = 3 cell culture wells). Two-way ANOVA with Bonferroni correction. f, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes by J774 macrophages in the absence of antibodies. Phagocytosis index normalized to control (SafeKO/SafeKO) cells. Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction.

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: CRISPR, Expressing, Phagocytosis Assay, Derivative Assay, Cell Culture, Incubation, Blocking Assay

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Phagocytosis assay for uptake of pHrodo-labelled Karpas-299 Cas9 cells expressing indicated sgRNAs by J774 macrophages in the presence or absence of anti-CD30 antibodies. Normalized phagocytosis index was calculated as average total pHrodo Red signal per well, normalized to signal in untreated control condition at final timepoint. Data represent mean +/− s.d. (n = 4 cell culture wells). Two-way ANOVA with Bonferroni correction. b, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes by human U937 macrophages in the presence or absence of anti-CD20 (rituximab) antibodies at indicated concentrations. Phagocytosis index normalized to control (SafeKO) Ramos cells without anti-CD20. Data represent mean +/− s.d. (n = 4 cell culture wells). Two-way ANOVA with Bonferroni correction. c, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells with indicated genotypes by human primary peripheral blood-derived macrophages, from two independent healthy de-identified human donors, in the presence or absence of 10 ng ml−1 anti-CD20 antibodies. Phagocytosis index normalized to control (SafeKO) Ramos cells without anti-CD20. Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction. d, Phagocytosis assay for uptake of pHrodo-labelled Ramos Cas9 cells expressing indicated sgRNAs by J774 macrophages with or without 24 h pre-treatment with 100 ng ml-1 LPS. Normalized phagocytosis index was calculated as average total pHrodo Red signal per well, normalized to signal in untreated control condition at final timepoint. Data represent mean +/− s.d. (n = 4 cell culture wells). e, Ramos Cas9 cells expressing indicated sgRNAs were incubated with Annexin V-FITC or anti-Calreticulin-DyLight-488 and analysed by flow cytometry. CRT, calreticulin. Data represent mean +/− s.d. (n = 3 independently stained samples). P-values were from two-tailed t-tests. f, Flow-cytometry based measurement of cell surface levels of CD20 in Ramos Cas9 cells expressing indicated sgRNAs. Data represent mean (n = 2 independently stained samples, except cells expressing CD20 sgRNA (n = 1)). g, Flow-cytometry based measurement of cell surface levels of CD47 in Ramos Cas9 cells expressing indicated sgRNAs. Data represent mean +/− s.d. (n = 3 independently stained samples). h, Flow-cytometry based measurement of cell surface levels of sialic acid in Ramos Cas9 cells expressing indicated sgRNAs. Where indicated, cells were treated with sialidase as a positive control. Data represent mean +/− s.d. (n = 3 independently stained samples). i, Viability assays (measured as cell confluence after 72 h on Incucyte, normalized to untreated SafeKO control cells) of indicated Ramos cells in the presence of indicated concentrations of 9 drugs. Data represent mean +/− s.d. (n = 3 cell culture wells). j, Flow-cytometry based measurement of forward scatter (FSC) and side scatter (SSC) in Ramos Cas9 cells expressing indicated sgRNAs. Data represent mean +/− s.d. (n = 3 independently analysed samples). k, Ramos-J774 adhesion assay in the presence of indicated antibody concentrations, using indicated GFP+ Ramos Cas9 knockout cells. Data represent mean +/− s.d. (n = 2 cell culture wells). l, Flow-cytometry based measurement of ADCP of Ramos Cas9 cells expressing indicated sgRNAs and stained with either calcein or CellTrace-Far-Red dye before incubation with J774 macrophages and anti-CD20 for 24h. Data represent mean +/− s.d. (n = 3 cell culture wells). Two-tailed t-tests were used to compare SafeKO and APMAPKO cells within each labeling condition.

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: Phagocytosis Assay, Expressing, Cell Culture, Derivative Assay, Incubation, Flow Cytometry, Staining, Two Tailed Test, Positive Control, Cell Adhesion Assay, Knock-Out, Labeling

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Levels of APMAP in ten cell lines measured by Western blot. All cell lines stably express Cas9 and were transduced with indicated sgRNAs. Actin served as loading control. Western blot to confirm knockout across all ten cell lines on one gel was performed once. For gel source data, see Supplementary Figure 1. b, Expression levels (TPM) of CD47 and APMAP in ten cell lines (data from CCLE). c, Survival measurements of selected (GFP+) cell lines in Fig. 3a, measured as percentage of GFP remaining after indicated number of hours of incubation with J774 macrophages in presence or absence of anti-CD47. Data represent mean +/− s.d. (n = 4 cell culture wells, except Karpas-299 (n = 3)). One-way ANOVA with multiple comparisons correction. d, Phagocytosis assays as in Fig. 3a, but with isotype control antibodies. Data represent mean +/− s.d. (n = 4 cell culture wells). One-way ANOVA with Bonferroni correction. e, Phagocytosis assay for uptake of pHrodo-labelled cells for indicated Cas9-expressing cell lines expressing indicated sgRNAs by J774 macrophages in the presence or absence of anti-EGFR/cetuximab antibodies. Phagocytosis index normalized to control (SafeKO) cells without anti-EGFR. Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction. f, Survival measurements of selected (GFP+) cell lines in Extended Data Fig. 6e, measured as percentage of GFP remaining after indicated number of hours of incubation with J774 macrophages in presence or absence of anti-EGFR. Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction. g, Representative photographs depicting Ramos tumours of indicated genotype extracted from NSG mice at 25 d following transplantation. h, SafeKO or APMAPKO Ramos cells were transplanted into NSG mice and allowed to form tumours. Mice were treated with anti-CD47 (B6H12, BioXCell) or PBS daily starting 17 d following transplantation, and tumour size was measured every 2 d. Data represent mean +/− s.e.m. (n = 5 (SafeKO groups) and 6 (APMAPKO groups)). Two-way ANOVA with Tukey correction (comparison between SafeKO/anti-CD47 and APMAPKO/anti-CD47 for final timepoint is shown). I, Mouse weights in Ramos (top) and NCI-H82 (bottom) xenograft experiments (Extended Data Fig. 6h, Fig. 3b). Data represent mean +/− s.d. Two-way ANOVA with Bonferroni correction (n = 5 (all NCI-H82 groups and Ramos SafeKO groups) and 6 (Ramos APMAPKO groups)). P-values are reported for the interaction between treatment groups. j, Single-cell suspensions were prepared from SafeKO or APMAPKO Ramos tumours treated with PBS or anti-CD20 (from experiment in Fig. 3c) and analysed for the presence of macrophages (CD45+/F4–80+/Cd11b+) as a percentage of all CD45+ cells. Gating strategy is shown (top/left). Data (bottom right) represent mean +/− s.e.m. (n = 6 (PBS groups) and 7 (antibody-treated groups)). One-way ANOVA with Tukey correction. k, Phagocytosis assay for uptake of pHrodo-labelled B16-F10 cells with indicated genotypes by J774 macrophages in the presence or absence of anti-TRP1 antibodies. Phagocytosis index normalized to control (SafeKO) cells without antibody. Data represent mean +/− s.d. (n = 4 cell culture wells). One-way ANOVA with Bonferroni correction. l, In vitro growth of B16-F10 cells of indicated genotypes, measured using time-lapse microscopy as total confluence per well over 6 d. Data represent mean +/− s.d. (n = 4 cell culture wells). m, SafeKO or APMAPKO B16-F10 cells were transplanted into syngeneic C57BL/6 mice and allowed to form tumours. Mice were treated with anti-TRP1 or mouse IgG2a isotype control antibody daily starting 5 d following transplantation, and tumour size was measured every 2 d. Data represent mean +/− s.e.m. (n = 7 for SafeKO groups, n = 6 for both APMAPKO groups). Two-way ANOVA with Tukey correction (comparison between SafeKO/anti-TRP1 and APMAPKO/anti-TRP1 for final timepoint is shown).

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: Western Blot, Stable Transfection, Transduction, Knock-Out, Expressing, Incubation, Cell Culture, Phagocytosis Assay, Transplantation Assay, In Vitro, Time-lapse Microscopy

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Schematic of macrophage screen, using a genome-wide CRISPR knockout library in J774 macrophages, for uptake of calcein+ SafeKO cells and far red+ APMAPKO Ramos cells. Req., required. b, Volcano plot of all genes required for uptake of APMAPKO cells (comparison 1 of screen depicted in a). c, Volcano plot of genes selectively required for uptake of APMAPKO cells relative to SafeKO cells (comparison 2 of screen depicted in a). d, Phagocytosis assay for uptake of pHrodo-labelled Ramos-Cas9 cells expressing indicated sgRNAs, incubated with J774 macrophages expressing indicated sgRNAs, in the presence of anti-CD20 antibodies, normalized to Safe-1 macrophages with SafeKO Ramos cells (n = 4 culture wells). P values are for comparisons with Safe-1 macrophages for each Ramos cell type. e, Phagocytosis assay for uptake of pHrodo-labelled SafeKO Ramos cells, incubated with U937 macrophages, anti-CD20 antibodies and indicated GPR84 agonists at indicated concentrations, normalized to untreated cells (n = 4 cell culture wells). P values are for comparisons with untreated cells. f, Model for APMAP-mediated regulation of macrophage phagocytosis through GPR84, GNB2 and PREX1. The role of PREX1 as a coincidence detector for Gβ and phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) in Rac activation is indicated; the Rac-GEF domain of PREX1 is synergistically released from an autoinhibited state upon binding of these two ligands at distinct sites. The question mark indicates a putative role of APMAP enzymatic activity in preventing GPR84-dependent phagocytosis activation. FcR, Fc receptor; mAb, monoclonal antibody. Dotted lines in b, c indicate 5% FDR. In d, e, data are mean ± s.d. One-way ANOVA with Bonferroni correction.

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: Genome Wide, CRISPR, Knock-Out, Phagocytosis Assay, Expressing, Incubation, Cell Culture, Activation Assay, Binding Assay, Activity Assay

Journal: Nature

Article Title: Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis

doi: 10.1038/s41586-021-03879-4

Figure Lengend Snippet: a, Phagocytosis assay for uptake of pHrodo-labelled Ramos Cas9 cells expressing Safe-targeting sgRNAs by J774 macrophages in the presence of anti-CD20 antibodies and indicated concentrations of GPR84 agonists. Data represent mean (n = 2 cell culture wells). b, Phagocytosis assay for uptake of pHrodo-labelled Ramos Cas9 cells expressing APMAP-targeting sgRNAs by J774 macrophages in the presence of anti-CD20 antibodies and indicated concentrations of GPR84 agonists. Data represent mean (n = 2 cell culture wells). c, Phagocytosis assay for uptake of pHrodo-labelled SafeKO Ramos Cas9 cells by J774 macrophages in the presence (left) or absence (right) of anti-CD20 antibodies and 100 μM saturated fatty acids of indicated carbon chain length (n = 2, acetic acid; n = 10, capric acid; n = 16, palmitic acid; n = 22, docosanoic acid). Data represent mean +/− s.d. (n = 4 cell culture wells). One-way ANOVA with Bonferroni correction. d, Heatmap of normalized phagocytosis index of Ramos cells incubated with U937 macrophages expressing indicated sgRNAs, in the presence of indicated concentrations of 6-OAU and anti-CD20. Data represent mean (n = 4 cell culture wells). e, Heatmap of normalized phagocytosis index of Ramos cells incubated with J774 macrophages expressing indicated sgRNAs, in the presence of indicated concentrations of 6-OAU and anti-CD47. Data represent mean (n = 4 cell culture wells). f, Phagocytosis assay for uptake of pHrodo-labelled Ramos Cas9 cells expressing Safe-targeting sgRNAs by J774 macrophages in the presence of anti-CD20 antibodies and GPR84 agonists (100 μM capric acid, 100 nM 6-OAU, 10 nM ZQ-16). Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction. P-values are for comparison to untreated condition for each macrophage genotype.

Article Snippet: The following antibodies were used: mouse monoclonal anti-APMAP (OTI4F6, Origene, 1:2,000 dilution), mouse monoclonal anti-Flag (clone M2, F1804, Sigma, 1:2,000 dilution), mouse monoclonal anti-GAPDH (clone 6C5, AM4300, Fisher, 1:5,000 dilution), and rabbit polyclonal anti-β-actin (ab8227, Abcam, 1:2,000 dilution).

Techniques: Phagocytosis Assay, Expressing, Cell Culture, Incubation