Journal: eLife

Article Title: Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging

doi: 10.7554/eLife.66749

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Satellite Cell Isolation Kit, mouse , Miltenyi Biotec , Miltenyi Biotec 130104268 , .

Techniques: Staining, Activity Assay, Blocking Assay, Plasmid Preparation, Recombinant, Membrane, Hybridization, Sequencing, Cell Isolation, In Situ, Software

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 3. CmAb-(IL10)2-Mediated Antitumor Effects Depend on Host Immunity (A and B) Tumor growth in C57BL/6J (A) or Rag1/ (B) mice (n = 5) bearing B16-cEGFR tumors treated by intratumoral (i.t.) injection of Cetuximab, CmAb-(IL10)2, or control IgG (indicated by arrows). (C) Quantification of OVA tetramer-positive (OVA-specific) CD8+ T cells in tumor tissues collected from B16-cEGFR-OVA tumor-bearing C57BL/6J mice (n = 4–5) treated twice by i.t. injection with control IgG or CmAb-(IL10)2 on days 11 and 14 after tumor cell inoculation. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (D) IFN-g ELISPOT assay of splenocytes collected from B16-cEGFR-OVA tumor-bearing C57BL/6J mice (n = 5–6) treated three times by i.t. injection of control IgG or CmAb-(IL10)2. The spleens were harvested 9 days after the first treatment. OT1 peptide, OVA-derived SIINFEKL peptide; SIY, a control peptide SIYRYYGL. (E) Tumor growth in C57BL/6J mice (n = 5) bearing B16-cEGFR tumors treated with control IgG or CmAb-(IL10)2 (i.t., indicated by arrows). a-CD8 or a-CD4 antibodies were administered for T cell depletion during the CmAb-(IL10)2 treatment. (F and G) Tumor growth in NSG-SGM3 (F) and NSG-SGM3 humanized (G) mice (n = 5) bearing A431 tumors treated with CmAb-(IL10)2 or Cetuximab on days 11, 14, 17, and 20 after tumor cell inoculation. (A–G) Data are shown as means ± SEM. **p < 0.01, ****p < 0.0001; ns, not significant. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Injection, Control, Cytometry, Enzyme-linked Immunospot, Derivative Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 4. DCs are Essential for the Antitumor Effects of CmAb-(IL10)2 by Preventing Apoptosis of Antigen-Specific CD8+ T Cells (A and B) Proliferation of CFSE-labeled CD8+ OT1 T cells co-cultured with BMDCs from C57BL/6J mice in the presence of OVA and treated with CmAb-(IL10)2, Cetuximab, or vehicle. The percentage (A) and the number (B) of proliferating CD8+T cells were assessed by flow cytometry at the indicated time points. (C) Apoptosis of proliferating CD8+ T cells at 72 h after co-culture as described in (A and B), assessed by flow cytometry.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Labeling, Cell Culture, Cytometry, Co-Culture Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 5. IL-10R Signaling on DCs Is Required for Preventing Apoptosis of Antigen-Specific CD8+ T Cells (A) Proliferation of CFSE-labeled CD8+ OT1 T cells co-cultured with BMDCs from WT (left) or Il10r/ (right) mice in the presence of OVA treated with CmAb-(IL10)2 or vehicle, assessed by flow cytometry at the indicated time points. (B) Cell number of proliferating CD8+ OT1 T cells co-cultured with BMDCs from Il10r/ mice in the presence of OVA and treated with CmAb-(IL10)2 or vehicle, assessed by flow cytometry at the indicated time points. (C) Apoptosis of proliferating CD8+ T cells at 72 h after co-culture as described in (B), assessed by flow cytometry. (D) Scheme of adoptive transfer of CD8+ T cells (2 3 104 OT1 CD8+ mixed with 2 3 106 WT CD8+ T cells) and CmAb-(IL10)2 treatment of Rag1/ or Il10r/ Rag1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Labeling, Cell Culture, Cytometry, Co-Culture Assay, Adoptive Transfer Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 6. CmAb-(IL10)2 Prevents Antigen-Specific CD8+ T Cell Apoptosis through Regulating DC-Mediated IFN-g Production (A) Apoptosis assessment of re-stimulated CD8+ T cells by co-culturing antigen-activated CD8+ OT1 T cells with BMDCs from C57BL/6J mice in the presence of OVA and CmAb-(IL10)2 or vehicle, determined at 48 h after re-stimulation by flow cytometry. (B) IFN-g production from the indicated co-cultures of DCs and CD8+ OT1 T cells in the presence of OVA and treated with CmAb-(IL10)2 or vehicle. (C and D) Apoptosis of proliferating CD8+ T cells co-cultured with BMDCs from WT (C) or Il10r/ (D) mice in the presence of OVA and treated with CmAb-(IL10)2, IFN-g, or a-IFN-g (10 mg/mL), assessed by flow cytometry. (E) Apoptosis of CD8+ T cells in B16-cEGFR-OVA tumor tissues from Rag1/ mice (n = 5–6) i.t. treated with 1 3 106 antigen-activated CD8+ OT1 T cells plus control IgG, CmAb-(IL10)2 or anti-IFN-g (150 mg, i.p.) on day 11 after tumor cell inoculation. Tumor tissues were collected 2 days after treatment and analyzed by flow cytometry. (F) Apoptosis of OVA tetramer-positive CD8+ T cells in B16-cEGFR-OVA tumor tissues from Ifng/ mice (n = 7) i.t. treated with control IgG or CmAb-(IL10)2 on days 8 and 11 after tumor cell inoculation. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (A–F) Data are shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Cytometry, Cell Culture, Control

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 7. CmAb-(IL10)2 Can Prevent Antigen-Specific CD8+ TIL Apoptosis and Improve the Antitumor Effects of Immune Checkpoint Blockade in the Treatment of Advanced Tumors (A) Apoptosis of OVA tetramer-positive CD8+ T cells in B16-cEGFR-OVA tumor tissues from C57BL/6J mice (n = 7) treated twice by a-PD-L1 and a-CTLA-4 (immune checkpoint blockade [ICB]) in combination with control IgG or CmAb-(IL10)2. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (B and C) Scheme (B) (top), tumor growth (B) (bottom), and survival curve (C) of the advanced B16cEGFR tumor-bearing (80–120 mm3) C57BL/6J mice (n = 6–7) treated with CmAb-(IL10)2, ICB, or the combination therapy as indicated. (D) Tumor growth after challenge with B16-cEGFR cells in treatment-naı¨ve mice or mice cured by the combination therapy for ICB and CmAb-(IL10)2. (E) Scheme of treatment (left) and tumor growth (right) of C57BL/6J mice (n = 5–7) bearing advanced B16-cEGFR tumors treated with CmAb-(IL10)2, ICB, or the combination therapy as indicated. (A–E) Data are shown as means ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Control, Cytometry

Journal: Cancer Research

Article Title: Slit2 Inhibits Breast Cancer Metastasis by Activating M1-Like Phagocytic and Antifibrotic Macrophages

doi: 10.1158/0008-5472.can-20-3909

Figure Lengend Snippet: Figure 5. Slit2 reduces the expression of IL6, thereby inhibiting TAMs activity. A, Total RNA of CD11bþ cells sorted from rSlit2-N PBS-treated MMTV-PyMT tumorswas subjected to gene expression analysis using NanoString technology. The heatmap shows differentially expressed top genes. B, Total RNA was isolated from MDA-MB-231 cells overexpressing Slit2 (231-Slit2) or vector control (231-vec) and analyzed for gene expression using microarray technology. The heatmap shows differentially expressed top genes. C, 231-Slit2 or 231-Vec cells CM was subjected to cytokine array analysis. The representative image shows differentially expressed molecules. D, The graph shows levels of IL6 in CM derived from 231-Sli2 or Vec CM detected by ELISA. E, Expression of Robo1 in MDA-MB-231 cells transduced with lentivirus expressing siRNA specific to Robo1 (si-Robo1) or control (si-ctrl) by Western blot. F, b-actin was used as loading control. MDA-MB-231 parental control, si-Robo1, or si-ctrl from E was treated with rSlit2-N or PBS and phosphorylation at p65 of NFkB (p-NFkB) or total NFkB (t-NFkB) was analyzed. G, Graph showing levels of IL6 in si-Robo1 or si-ctrl MDA-MB-231 cells treated with rSlit2-N or PBS detected by ELISA (n ¼ 3 each group). H, BMDMs were pretreated with serum-free CM (CTRL), or 231-Slit2 CM or 231-Vec CM or 231-Vec CM preincubated with IL6 nAb (231-Vec þ IL6nAb) or IL6 nAb. After 2 hours, pH-Rhodo–labeled S. Aureus particles were added to the cells and recombinant IL6 was added to the 231-Slit2 CM cells (231-Slit2-rIL6). The kinetics of S. Aureus phagocytosis was analyzed using a fluorescence plate reader at every 15 minutes up to 4 hours. , P < 0.05; , P < 0.01; , P < 0.001 using Student t test.

Article Snippet: The cells were pretreated with mouse rSlit2-N (100 ng/mL) or IL6 neutralization antibody (BioXCell# BE0046) for 2 hours.

Techniques: Expressing, Activity Assay, Gene Expression, Isolation, Plasmid Preparation, Control, Microarray, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, Phospho-proteomics, Labeling, Recombinant