Figure S5 B. The expression of Siglec-7 on NK cells from donor 1 and donor 2 was analyzed. (B) Cytotoxicity assay using co-culture of DLD-1 or ST transfectants with PBMCs. Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity released into the medium from injured cells. They were measured using the LDH release detection kit. E:T refers to the ratio of effector cells (PBMCs) and target cells (DLD-1 and ST transfectants). Error bars indicate SEM. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, using one-way ANOVA. E:T = 100:1 p = 0.0003, E:T = 50:1 p = 0.0026, E:T = 25:1 p = 0.0166, E:T = 12.5:1 p = 0.0241. Tukey’s multiple comparison test, E:T = 100:1 DLD-1 vs. C3, p = 0.0129, DLD-1 vs. C4, p = 0.0059, DLD-1 vs. C8, p = 0.0002, E:T = 50:1 DLD-1 vs. C8, p = 0.0012, E:T = 25:1 DLD-1 vs. C8, p = 0.0346, E:T = 12.5:1 DLD-1 vs. C8, p = 0.0223, E:T = 6.25:1 DLD-1 vs. C8, p = 0.0314. Data are mean ± SD, n = 7 biological replicates. (C) PBMCs were co-cultured with DLD at E:T = 100:1 after prior treatment with 10 μg/mL anti-Siglec-7 antibody or isotype control antibody. The cytotoxic activity was then assessed with the LDH release detection kit. DLD-1 Control IgG vs. DLD-1 Anti-Sig-7, p = 0.2328, C3 Control IgG vs. C3 Anti-Sig-7, p = 0.0404, C4 Control IgG vs. C4 Anti-Sig-7, p = 0.0260, C8 Control IgG vs. C8 Anti-Sig-7, p = 0.0062, Data are mean ± SD, n = 4 biological replicates. (D and E) Co-culture of an NK cell line KHYG-1-Siglec-7 high (
Figure S3 B) and DLD-1 or ST8Sia6-transfectant C8. DLD-1 and C8 were transiently transfected with a red fluoresceine protein gene for labeling of the cells. The medium contained DAPI, which stains dead cells. The magnified image in the upper right of the panel shows dead cells stained with DAPI. Dead KHYG-1-Siglec-7 high stained using DAPI are indicated with blue arrows. Time-lapse imaging of co-cultured DLD-1 or C8 and KHYG-1-Siglec-7 high was acquired for 10 h . Scale bar indicates 50 μm. (F) Assessment of NK apoptosis in PBMCs co-cultured with DLD; PBMCs were stained with anti-CD3-FITC and anti-CD16/CD56-PE after co-culturing. Further staining with Annexin-V-APC and 7-AAD comparing Annexin-V and 7-AAD co-positive populations in Gate:A (Appendix Gating Information). (G) U937-Siglec-7 high were co-cultured with DLD-1 or ST transfectants for 10 min at 37°C. The U937 cells were collected and lysed. Immunoprecipitation was performed using an anti-Siglec-7 antibody and subsequent immunoblotting with an anti-phospho-tyrosine antibody (4G10), anti-Siglec-7 antibody, or anti-SHP-1. (H) Immunoprecipitation using anti-SHP-1 antibody and subsequent IB using anti-phosphotyrosine antibody (PY20) or anti-SHP-1 were performed. Phosphorylated Siglec-7 and SHP-1 were quantitated using ImageJ software (NIH) and the ratios of p-protein/total proteins in (G) and (H) are presented in (I) and (J), respectively. (I and J) Summary of phosphorylation ratios for Siglec-7 ((I) p-Siglec-7/Siglec-7) and SHP-1 ((J) p-SHP-1/SHP-1). DLD-1 and ST transfectants vs. (A and B) PBMCs, (C) KHYG-1-Siglec-7 high and (D–F) U937-Siglec-7 high . " width="100%" height="100%">
Journal: iScience
Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance
doi: 10.1016/j.isci.2024.111139
Figure Lengend Snippet: Siglec-7 ligand O-glycans suppress NK cell cytotoxity via inhibitory signal activation of ITIM/ITSM (A) The gating information for picking up NK cells from PBMCs is in Figure S5 B. The expression of Siglec-7 on NK cells from donor 1 and donor 2 was analyzed. (B) Cytotoxicity assay using co-culture of DLD-1 or ST transfectants with PBMCs. Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity released into the medium from injured cells. They were measured using the LDH release detection kit. E:T refers to the ratio of effector cells (PBMCs) and target cells (DLD-1 and ST transfectants). Error bars indicate SEM. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, using one-way ANOVA. E:T = 100:1 p = 0.0003, E:T = 50:1 p = 0.0026, E:T = 25:1 p = 0.0166, E:T = 12.5:1 p = 0.0241. Tukey’s multiple comparison test, E:T = 100:1 DLD-1 vs. C3, p = 0.0129, DLD-1 vs. C4, p = 0.0059, DLD-1 vs. C8, p = 0.0002, E:T = 50:1 DLD-1 vs. C8, p = 0.0012, E:T = 25:1 DLD-1 vs. C8, p = 0.0346, E:T = 12.5:1 DLD-1 vs. C8, p = 0.0223, E:T = 6.25:1 DLD-1 vs. C8, p = 0.0314. Data are mean ± SD, n = 7 biological replicates. (C) PBMCs were co-cultured with DLD at E:T = 100:1 after prior treatment with 10 μg/mL anti-Siglec-7 antibody or isotype control antibody. The cytotoxic activity was then assessed with the LDH release detection kit. DLD-1 Control IgG vs. DLD-1 Anti-Sig-7, p = 0.2328, C3 Control IgG vs. C3 Anti-Sig-7, p = 0.0404, C4 Control IgG vs. C4 Anti-Sig-7, p = 0.0260, C8 Control IgG vs. C8 Anti-Sig-7, p = 0.0062, Data are mean ± SD, n = 4 biological replicates. (D and E) Co-culture of an NK cell line KHYG-1-Siglec-7 high ( Figure S3 B) and DLD-1 or ST8Sia6-transfectant C8. DLD-1 and C8 were transiently transfected with a red fluoresceine protein gene for labeling of the cells. The medium contained DAPI, which stains dead cells. The magnified image in the upper right of the panel shows dead cells stained with DAPI. Dead KHYG-1-Siglec-7 high stained using DAPI are indicated with blue arrows. Time-lapse imaging of co-cultured DLD-1 or C8 and KHYG-1-Siglec-7 high was acquired for 10 h . Scale bar indicates 50 μm. (F) Assessment of NK apoptosis in PBMCs co-cultured with DLD; PBMCs were stained with anti-CD3-FITC and anti-CD16/CD56-PE after co-culturing. Further staining with Annexin-V-APC and 7-AAD comparing Annexin-V and 7-AAD co-positive populations in Gate:A (Appendix Gating Information). (G) U937-Siglec-7 high were co-cultured with DLD-1 or ST transfectants for 10 min at 37°C. The U937 cells were collected and lysed. Immunoprecipitation was performed using an anti-Siglec-7 antibody and subsequent immunoblotting with an anti-phospho-tyrosine antibody (4G10), anti-Siglec-7 antibody, or anti-SHP-1. (H) Immunoprecipitation using anti-SHP-1 antibody and subsequent IB using anti-phosphotyrosine antibody (PY20) or anti-SHP-1 were performed. Phosphorylated Siglec-7 and SHP-1 were quantitated using ImageJ software (NIH) and the ratios of p-protein/total proteins in (G) and (H) are presented in (I) and (J), respectively. (I and J) Summary of phosphorylation ratios for Siglec-7 ((I) p-Siglec-7/Siglec-7) and SHP-1 ((J) p-SHP-1/SHP-1). DLD-1 and ST transfectants vs. (A and B) PBMCs, (C) KHYG-1-Siglec-7 high and (D–F) U937-Siglec-7 high .
Article Snippet: Anti-human CD3 PE/Cy7 , Thermo Fisher Scientific , Cat# 25-0037-42, RRID: AB_2573326.
Techniques: Activation Assay, Expressing, Cytotoxicity Assay, Co-Culture Assay, Activity Assay, Comparison, Cell Culture, Control, Transfection, Labeling, Staining, Imaging, Immunoprecipitation, Western Blot, Software, Phospho-proteomics
Journal: iScience
Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance
doi: 10.1016/j.isci.2024.111139
Figure Lengend Snippet:
Article Snippet: Anti-human CD3 PE/Cy7 , Thermo Fisher Scientific , Cat# 25-0037-42, RRID: AB_2573326.
Techniques: Blocking Assay, Plasmid Preparation, Purification, Control, Staining, Recombinant, Membrane, Mutagenesis, Cytotoxicity Assay, Cloning, Expressing, Sequencing, Software
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