Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a , b The expression of glycolysis-related genes was detected by RT-qPCR in both B7-H3-overexpressing HCT116 ( a ) and RKO ( b ) cells. c , d The mRNA level of HK2 was detected by RT-qPCR in both HCT116 ( c ) and RKO ( d ) cells after transfection with siRNA NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. e HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both HCT116 and RKO cells after transfection with NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. β-actin served as a loading control. f HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. β-actin served as a loading control. g Schematic representation of the proposed B7-H3/STAT3/HK2 axis. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Western Blot, Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a The protein level of HK2 in B7-H3-overexpressing HCT116 or RKO cells after transfection with siRNA negative control (NC) or HK2 siRNA transfection were analyzed by western blot. β-actin served as a loading control. b , c Glucose consumption ( b ) and lactate production ( c ) were measured in B7-H3-overexpressing HCT116 or RKO cells transfected with NC or HK2 siRNA. d , e Glucose consumption ( d ) and lactate production ( e ) were measured in B7-H3-overexpressing HCT116 or RKO cells treated with PBS or 2-DG. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Transfection, Negative Control, Western Blot, Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a , b Colony formation assay of B7-H3-overexpressing HCT116 or RKO cells exposed to L-OHP. The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. c , d Cell viability was analyzed using the CCK-8 kit. B7-H3-overexpressing HCT116 or RKO cells showed resistance to L-OHP ( c ) or 5-FU ( d ). The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. e B7-H3-overexpressing HCT116 or RKO cells showed less cell apoptosis than control cells (EV) after L-OHP treatment. The effect of B7-H3 on cell apoptosis was abolished by HK2 siRNA or 2-DG. f The protein levels of Bcl-2 and Bax were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. The effect of B7-H3 on Bcl-2 and Bax expression was abolished by HK2 siRNA. β-actin served as a loading control. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Colony Assay, CCK-8 Assay, Control, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a Images of IHC analysis of B7-H3 and HK2 protein expression and hematoxylin and eosin (H&E) staining of CRC ( n = 126) tissue sections. One representative image is shown. b , c B7-H3 ( b ) and HK2 ( c ) protein expression based on their staining index in nonmalignant adjacent tissues (NAT) and CRC specimens. d Correlation analysis of the staining index of expression levels of B7-H3 and HK2 protein in human CRC specimens ( n = 126). e , f B7-H3 ( e ) and HK2 ( f ) protein expression based on their staining index in CRC specimens at different clinical stages. Values are expressed as means (SEMs). * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Expressing, Staining

Journal: Cancer Immunology, Immunotherapy

Article Title: The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions

doi: 10.1007/s00262-018-2160-x

Figure Lengend Snippet: BGB-A317/IgG4 S228P mediates crosslinking between PD-1 and FcγRI receptors determined by biochemical and cell signaling assays. a Overlaid sensorgrams of BGB-A317 and BGB-A317/IgG4 S228P binding to PD-1 followed by binding with FcγRI. The PD-1 antibody (either BGB-A317 or BGB-A317/IgG4 S228P ) was injected onto the surface of human PD-1/His-coated CM5 chip, followed by FcγRI association and dissociation. The arrows indicated the time points when the analytes were injected. b Cartoon interpretation of the SPR assay in ( a ) showing that BGB-A317/IgG4 S228P mediates crosslinking between PD-1 and FcγRI, while BGB-A317 does not. c Comparison of PD-1 antibody functional activities by P3Z assay. In the assays, detection of IL-2 secretion served as a quantitative indicator of PD-1 signaling. The dose–response was assayed either in the “two-cell line” co-cultures (HuT78/P3Z and HEK293/PD-L1) presented by solid symbols or in the “three-cell line” system (HuT78/P3Z, HEK293/PD-L1 and FcγRI + THP-1 cells) presented by open symbols. Ab conc . Antibody concentration. d Rabbit Anti-CD64 (FcγRIα) polyclonal antibody (pAb) restored the inhibitory effect of BGB-A317/IgG4 S228P on PD-1-signaling in the “three-cell” co-culture system. THP-1 cells were pre-treated with the rabbit Anti-CD64 pAb before co-culturing with HuT78/P3Z and HEK293/PD-L1 cells in the presence of BGB-A317/IgG4 S228P . The placebo contained antibody buffer solution only

Article Snippet: Then, the sections were blocked by 3% bovine serum albumin in PBS for 1 h and 0.3% H 2 O 2 solution in PBS for 10 min, and afterwards, stained by the antibodies against human CD8 (SP16, ZSGB-Bio), PD-1 (NAT105, Abcam), CD64 (3D3, Abcam), and mouse CD64 (Clone 027, Sino Biological) at 4 °C overnight.

Techniques: Binding Assay, Injection, SPR Assay, Functional Assay, Concentration Assay, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions

doi: 10.1007/s00262-018-2160-x

Figure Lengend Snippet: BGB-A317/IgG4 S228P induces primary M2 macrophages to phagocytose PD-1 + T cells (ADCP) and to express IL-10 gene. a CD64 (FcγRI) and CD32 (FcγRII) expression on the in vitro differentiated M2 macrophages as determined by FACS. b ADCP assay using M2 macrophages. Primary M2 macrophages were co-cultured with CFSE-labeled PD-1 + HuT78/PD-1 cells overnight in the presence of the indicated antibodies. HuIgG was used as negative control. Representative dot plots of three independent experiments are shown. M2 Mac: M2 macrophage. c Bar graphs summarized the results of three independent experiments. The % of ADCP was determined as described in the “ ”. The mean + SD represents triplicate data points. ## P < 0.01, comparing BGB-A317/IgG4 S228P versus huIgG. ** P < 0.01, comparing BGB-A317/IgG4 S228P versus BGB-A317. d Rabbit anti-CD64 polyclonal antibody neutralizes FcγRI-mediated ADCP. Rabbit IgG and placebo were used as negative controls. e Crosslinking of FcγRI + M2 macrophages to plate-coated PD-1 by BGB-A317/IgG4 S228P induces IL-10 gene expression. M2 macrophages were added to PD-1-coated 96-well plates in the presence of anti-PD-1 Abs and cultured overnight. The IL-10 gene expression was assayed by real-time PCR. IL-10 expression in huIgG-treated M2 macrophages was set as a baseline. The mRNA levels of placebo (antibody buffer solution), BGB-A317 or BGB-A317/IgG4 S228P -treated M2 macrophages were normalized against the baseline. The results from three independent experiments are shown as mean + SD of duplicate data points. HuIgG is a mixture of human IgG1, IgG2, IgG3 and IgG4 (Invitrogen)

Article Snippet: Then, the sections were blocked by 3% bovine serum albumin in PBS for 1 h and 0.3% H 2 O 2 solution in PBS for 10 min, and afterwards, stained by the antibodies against human CD8 (SP16, ZSGB-Bio), PD-1 (NAT105, Abcam), CD64 (3D3, Abcam), and mouse CD64 (Clone 027, Sino Biological) at 4 °C overnight.

Techniques: Expressing, In Vitro, Cell Culture, Labeling, Negative Control, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy

Article Title: The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions

doi: 10.1007/s00262-018-2160-x

Figure Lengend Snippet: Crosslinking of T cells and FcγRI positive cells in vivo. a Representative low power (40×) images of multiplex immunofluorescence staining of whole mount tumors treated with BGB-A317 or BGB-A317/IgG4 S228P . The xenograft tumor model was generated by subcutaneously implanting the allogenic human epidermoid cancer cells (A341 cell line) and PBMCs in NOD/SCID mice. Representative high power images (200×) were shown in ( b ). The biomarkers are indicated by green (hCD8), red (mCD64) and blue (nucleus), respectively. c – e Quantified results of hCD8, mCD64 staining intensity and co-localization of hCD8 and mCD64 in each indicated group ( n = 5). Six to ten images in each tumor sample were captured by Vectra, and the staining intensity and co-localization was quantified using inForm software. (Note: CD64 = FcγRIα)

Article Snippet: Then, the sections were blocked by 3% bovine serum albumin in PBS for 1 h and 0.3% H 2 O 2 solution in PBS for 10 min, and afterwards, stained by the antibodies against human CD8 (SP16, ZSGB-Bio), PD-1 (NAT105, Abcam), CD64 (3D3, Abcam), and mouse CD64 (Clone 027, Sino Biological) at 4 °C overnight.

Techniques: In Vivo, Multiplex Assay, Immunofluorescence, Staining, Generated, Software

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: Mouse Anti-PODXL (3D3) , Santa Cruz Biotechnology , Cat# sc-23904, RRID: AB_2166006.

Techniques: Blocking Assay, Plasmid Preparation, Purification, Control, Staining, Recombinant, Membrane, Mutagenesis, Cytotoxicity Assay, Cloning, Expressing, Sequencing, Software