Journal: Current biology : CB

Article Title: SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors

doi: 10.1016/j.cub.2017.06.005

Figure Lengend Snippet: (A) Marrow macrophages were engineered and mixed with streptavidin beads of 900 nm to 6.7 μm diameter or else with disaggregated tdTom A549 tumors. Beads are opsonized with anti-streptavidin except for a non-targeted control in which SIRPα-inhibited macrophages were incubated with 2.1 μm lacking anti-streptavidin. These were not engulfed. After pre-incubation of the mixtures for up to 150 min, mixtures were plated for 24 hr on top of 5-μm pore transwells. Cells collected from transwell tops and bottoms were stained for CD11b+,F4/80+ and analyzed by flow cytometry for engulfment (STAR Methods).

Article Snippet: REAGENT OR RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human SIRPα (clone SE7C2) Santa Cruz sc-23863 Rat anti-mouse SIRPα (clone P84) BD Biosciences 552371 Rabbit anti-human RBCs (polyclonal) Rockland 109-4139 Anti-human CD41-FITC (HIP8) Biolegend 303703 Anti-human CD47-FITC (Clone CC2C6) Biolegend 323106 Rat Anti-human CD47 (Clone B6H12) BD Biosciences 556044 Anti-mouse CD11b-PE/Cy7 (Clone M1/70) Biolegend 101215 Anti-mouse F4/80-APC/Cy7 (clone BM8) Biolegend 123113 Donkey Anti-rabbit AF488 ThermoFisher A-21206 Donkey Anti-rabbit AF647 ThermoFisher A-31573 IgG from rabbit serum Sigma I5006-10MG Mouse anti-human Mucin (Clone S.854.6) ThermoFisher MA5-15131 Cetuximab InVivogen hegfr-mab1 Goat anti-rabbit AF700 ThermoFisher A-21038 Goat anti-rat AF647 ThermoFisher A-21247 Anti-mouse Ly-6G (Gr-1) APC (Clone RB6-8C5) Biolegend 108411 Anti-mouse CD135-BV421 (Clone A2F10.1) Biosciences 562898 Anti-mouse SIRPα-FITC (Clone P84) Biosciences 144005 Anti-human CD14-BV421 (Clone HCD14) Biolegend 325627 Anti-human CD33-APC (Clone WM53) Biolegend 303407 Anti-human CD66-PerCP/Cy5.5 (Clone G10F5) Biolegend 305107 Biological Samples Human Bone Marrow AllCells ABM001-1 NSG Mice The Jackson Laboratory 5557 Chemicals, Peptides, and Recombinant Proteins 7-Amino-actinomycin D Sigma A9400-1MG Hoechst 33342 ThermoFisher H3570 Phorbol myristate acetate Sigma P8139-1MG CFDA-SE ThermoFisher V12883 RBC Lysis Buffer Sigma R7757 Dispase StemCell Technologies 7913 Collagenase Sigma C0130-100MG DNase I Sigma 11284932001 Anti-streptavidin (polyclonal) Sigma S6390 Critical Commercial Assays F(Ab′)2 Preparation Kit ThermoFisher 44988 SIRPα ShRNA Lentiviral Plasmid Santa Cruz sc-44106-SH Streptavidin Beads ThermoFisher 11205D RNeasy plus Mini Kit QIAGEN 74134 TruSeq Stranded mRNA Library Prep kit Illumina RS-122-2101 Cell Lines Human adenocarcinoma (A549) ATCC CCL-185 Human Monocyte (THP-1) ATCC TIB-202 Primary liver carcinoma (EC4) Chi Van Dang’s Lab N/A Mesenchymal Stem Cells Progeria Research Foundation N/A Human hepatocellular carcinoma (HepG2) ATCC HB-8065 Mouse myoblast (C2C12) ATCC CRL-1772 Software MaxQuant Version 1.5.3.8 Max Planck Institute of Biochemistry http://www.biochem.mpg.de/5111795/maxquant Living Image Perkin Elmer http://www.perkinelmer.com/product/spectrum-200-living-image-v4series-1-128113 ImageJ NIH https://imagej.nih.gov/ij/ Open in a separate window KEY RESOURCES TABLE

Techniques: Incubation, Staining, Flow Cytometry

Journal: Current biology : CB

Article Title: SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors

doi: 10.1016/j.cub.2017.06.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT OR RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human SIRPα (clone SE7C2) Santa Cruz sc-23863 Rat anti-mouse SIRPα (clone P84) BD Biosciences 552371 Rabbit anti-human RBCs (polyclonal) Rockland 109-4139 Anti-human CD41-FITC (HIP8) Biolegend 303703 Anti-human CD47-FITC (Clone CC2C6) Biolegend 323106 Rat Anti-human CD47 (Clone B6H12) BD Biosciences 556044 Anti-mouse CD11b-PE/Cy7 (Clone M1/70) Biolegend 101215 Anti-mouse F4/80-APC/Cy7 (clone BM8) Biolegend 123113 Donkey Anti-rabbit AF488 ThermoFisher A-21206 Donkey Anti-rabbit AF647 ThermoFisher A-31573 IgG from rabbit serum Sigma I5006-10MG Mouse anti-human Mucin (Clone S.854.6) ThermoFisher MA5-15131 Cetuximab InVivogen hegfr-mab1 Goat anti-rabbit AF700 ThermoFisher A-21038 Goat anti-rat AF647 ThermoFisher A-21247 Anti-mouse Ly-6G (Gr-1) APC (Clone RB6-8C5) Biolegend 108411 Anti-mouse CD135-BV421 (Clone A2F10.1) Biosciences 562898 Anti-mouse SIRPα-FITC (Clone P84) Biosciences 144005 Anti-human CD14-BV421 (Clone HCD14) Biolegend 325627 Anti-human CD33-APC (Clone WM53) Biolegend 303407 Anti-human CD66-PerCP/Cy5.5 (Clone G10F5) Biolegend 305107 Biological Samples Human Bone Marrow AllCells ABM001-1 NSG Mice The Jackson Laboratory 5557 Chemicals, Peptides, and Recombinant Proteins 7-Amino-actinomycin D Sigma A9400-1MG Hoechst 33342 ThermoFisher H3570 Phorbol myristate acetate Sigma P8139-1MG CFDA-SE ThermoFisher V12883 RBC Lysis Buffer Sigma R7757 Dispase StemCell Technologies 7913 Collagenase Sigma C0130-100MG DNase I Sigma 11284932001 Anti-streptavidin (polyclonal) Sigma S6390 Critical Commercial Assays F(Ab′)2 Preparation Kit ThermoFisher 44988 SIRPα ShRNA Lentiviral Plasmid Santa Cruz sc-44106-SH Streptavidin Beads ThermoFisher 11205D RNeasy plus Mini Kit QIAGEN 74134 TruSeq Stranded mRNA Library Prep kit Illumina RS-122-2101 Cell Lines Human adenocarcinoma (A549) ATCC CCL-185 Human Monocyte (THP-1) ATCC TIB-202 Primary liver carcinoma (EC4) Chi Van Dang’s Lab N/A Mesenchymal Stem Cells Progeria Research Foundation N/A Human hepatocellular carcinoma (HepG2) ATCC HB-8065 Mouse myoblast (C2C12) ATCC CRL-1772 Software MaxQuant Version 1.5.3.8 Max Planck Institute of Biochemistry http://www.biochem.mpg.de/5111795/maxquant Living Image Perkin Elmer http://www.perkinelmer.com/product/spectrum-200-living-image-v4series-1-128113 ImageJ NIH https://imagej.nih.gov/ij/ Open in a separate window KEY RESOURCES TABLE

Techniques: Recombinant, Lysis, shRNA, Plasmid Preparation, Software

Journal: Current biology : CB

Article Title: SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors

doi: 10.1016/j.cub.2017.06.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-human SIRPα (clone SE7C2) , Santa Cruz , sc-23863.

Techniques: Recombinant, Lysis, shRNA, Plasmid Preparation, Software

Journal: Experimental Biology and Medicine

Article Title: Immune regulation effect of lienal polypeptides extract in Lewis lung carcinoma-bearing mice treated with cyclophosphamide

doi: 10.1177/1535370217737982

Figure Lengend Snippet: LP enhanced the protein expressions of CD47/SIRPα/SHP-1 in tumor tissues of LLC-bearing mice. CTX, LP, and TP5 were, respectively, administered to mice by intraperitoneal injection once daily from the sixth day after subcutaneous inoculation of LLC cells and lasted for 15 days. (a) The protein expressions of CD47/SIRPα/SHP-1 in tumor tissue were analyzed by Western blot and (b–d) their corresponding densities were quantified. Results were presented as mean ± SEM. The data were analyzed by ANOVA and a Tukey post hoc test using GraphPad Prism 6. **P < 0.01 and *P < 0.05 versus control group, ##P < 0.01 and #P < 0.05 versus CTX group were considered statistically significant. ANOVA: analysis of variance; CTX: cyclophosphamide; LLC: Lewis lung carcinoma; LP: lienal polypeptide; SEM: standard error of mean; SHP-1: Src homology phosphatase-1; SIRPα: signal regulatory protein α. (A color version of this figure is available in the online journal.)

Article Snippet: Anti-CD47 antibody and Anti-Src homology phosphatase-1 (SHP-1) antibody were brought from Abcam (Cambridge, MA, USA), Anti-signal regulatory protein α (SIRPα) antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), β-actin was bought from FuDe Biological Technology Co., Ltd (Hangzhou, China).

Techniques: Injection, Western Blot