engineered human embryonic kidney cell line hek lucia rig  (InvivoGen)

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: Effect of S15 overexpression on survival in PDAC and NSCLC patients and detection of S15 in tumor tissues . (A) S15 is overexpressed in PDAC patients, as shown by data from TCGA. (B-C) S15 is also overexpressed in NSCLC patients, including those with metastatic disease (data from TCGA). (D) Kaplan-Meier survival analysis indicates that pancreatic cancer patients with high S15 expression exhibit significantly lower overall survival (data from TCGA). (E-G) S15 expression was detected in pancreatic cancer patient tumor tissues by immunohistochemistry (IHC) staining. Primary anti-human S15 antibody was used at a 1:500 dilution (0.9 mg/mL), and secondary rabbit anti-human Alexa594 antibody was used at a 1:1000 dilution for detection. (H-J) S15 expression was also detected in NSCLC patient tumor tissues using the same staining protocol with anti-human S15 and rabbit anti-human Alexa594 antibodies.

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: Over Expression, Expressing, Immunohistochemistry, Staining

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: Characterization of STAB in vitro . (A) Schematic representation of the STAB molecule in the IgG-scFv format that binds CD3 and S15. (B) SDS-PAGE gel analysis confirms the expression of the STAB construct, showing correctly sized heavy and light chains. (C) Binding affinity of STAB to S15 compared to the parent S15 mAb, measured by ELISA. (D) Binding affinity of STAB to CD3 compared to the parent CD3 mAb, measured by ELISA. (E-F) Representative flow cytometry histograms demonstrating proliferation of T cells induced by (E) control anti-human IgG and (F) STAB, assessed using the CFSE proliferation assay. PBMCs from different donors were stained with 5 μg CFSE. After staining, PBMCs were cultured with either control IgG or STAB for 3 days, followed by harvesting and preparation of single-cell suspensions, and finally analysis by flow cytometry. (G) Quantification of fraction of CD3⁺ T cells following control IgG or STAB treatment. Data are presented as the mean ± standard error of the mean (SEM). ***p < 0.001.

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: In Vitro, SDS Page, Expressing, Construct, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Proliferation Assay, Staining, Cell Culture

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: STAB enhances T cell-mediated killing of S15+ human Panc-1 PDAC cells and human H460 NSCLC cells in vitro . PBMCs from different donors were stained with 5 μg CFSE, then cultured with tumor cells in presence of either Control IgG or STAB for 3 days, followed by harvesting and preparation of single-cell suspensions. CD3+CD45+ cell and tumor cell populations were analyzed by flow cytometry (n = 4). (A-B) Representative flow cytometry gating strategy for CD3⁺CD45⁺ T cells in Panc-1 co-culture. (C) Quantification of CD3⁺ T cells and GFP⁺ Panc-1 cells after treatment with Control IgG or STAB. (D-E) Representative flow cytometry gating strategy for GFP⁺ Panc-1 cells. (F) Luminescence-based assay results from Panc-1 co-culture with PBMCs treated with STAB or Control IgG, showing STAB-mediated tumor cell killing. (G-H) Representative flow cytometry gating strategy for CD3⁺CD45+ T cells in H460 co-culture. (I) Quantification of CD3⁺ T cells and mCherry⁺ H460 cells after treatment with Control IgG or STAB. (J-K) Representative flow cytometry gating strategy for mCherry⁺ H460 cells. (L) Luminescence-based assay results from H460 co-culture with PBMCs treated with STAB or Control IgG, showing STAB-mediated tumor cell killing. Percentage of cells in the STAB-treatment group is presented relative to the Control Ab at an effector-to-target (E:T) ratio of 3:1. Statistical differences in cell killing between STAB and control IgG were assessed using two-way ANOVA and are denoted as * (*p < 0.05; **p < 0.01).

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: In Vitro, Staining, Cell Culture, Control, Flow Cytometry, Co-Culture Assay, Luminescence Assay

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: STAB effectively inhibits Panc-1 tumor growth and significantly prolongs survival compared to treatments with anti-S15 or anti-CD3 mAbs. (A&D) Inhibition of Panc-1 tumor growth in NSG mice. Panc-1 cells (1 × 10⁶ in 50 μL PBS) were injected subcutaneously, and tumor size was measured every 2 days using calipers. Panels A and D represent two independent experiments (1st round: n = 5; 2nd round: n = 6-9). (B&E) Kaplan-Meier survival curves for the mice in (A) & (D) . Survival was defined by humane endpoints or tumor size exceeding 1000 mm³. Each group comprised 6-9 mice. (C) Representative images of tumors from the control and STAB treatment groups at the study endpoint. (F) Tumor weights at the study endpoint. (G-J) Quantification of CD3⁺ T cells and activated Ki67⁺CD3⁺ T cells in peripheral blood at 12 and 16 days post-treatment. Blood (60 μL) was collected via submandibular bleeding, stained with CD3, CD45, and Ki67, and analyzed by flow cytometry. Statistical differences between STAB treatment and control anti-human IgG groups were assessed using one-way ANOVA. Results are denoted as * (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: Inhibition, Injection, Control, Staining, Flow Cytometry

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: STAB treatment increases T cell infiltration in tumors and reduces tumor-associated fibroblasts in desmoplastic Panc-1 tumors. Tumors were harvested at the study endpoint from different treatment groups, fixed in 10% formalin overnight, and then stored in 70% ethanol before being embedded in paraffin. (A-D) Tumor sections were stained with anti-CD3 (Alexa 594, red) and anti-S15 (Alexa 488, green) to evaluate T cell infiltration and S15 expression. Negative staining (without primary antibody or without secondary antibody) and control staining were performed. (E-G) Tumor sections were stained with anti-αSMA (Alexa 488, green) and DAPI (blue) to assess tumor-associated fibroblasts. Negative staining and control staining were also performed. (H) Bar graph showing quantification of αSMA staining as an indicator of fibroblast presence.

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: Staining, Expressing, Negative Staining, Control

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: The ZIKV NS5 protein inhibits RNA-triggered RIG-I-mediated IFN production, and this effect is enhanced by autophagy inhibition. ( a ) Quantitative assay of the inhibitory effect of NS5 on RNA-triggered RIG-I-mediated IFN production in HEK-Lucia TM RIG-I cells expressing NS5 (1.5 µg cDNA) and treated with 3p-hpRNA (100 nM), an RNA-specific RIG-I agonist, for 24 h before the IFN-I response was quantified by QUANTI-Luc. The data obtained from control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) and treated with 3p-hpRNA (100 nM) are shown. The effect of 3-MA (5 mM) on the NS5-mediated inhibition of 3p-hpRNA-triggered RIG-I-mediated IFN production is shown. The data are presented as the means ± S.E.M. of two independent experiments ( n = 6). ( b ) Quantitative Western blot analysis of the effect of 3-MA treatment on HEK-Lucia cells overexpressing NS5-myc (1.5 µg cDNA). This biochemical analysis corresponds to the cells shown in panel ( a ) for RNA-mediated RIG-I IFN production. Lanes 1 and 2 represent control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) and treated with PBS (vehicle for 3-MA) or 3-MA (5 mM), respectively. Lanes 3 and 4 represent cells overexpressing NS5 (NS5-HA plasmid, 1.5 µg cDNA) and treated with PBS or 3-MA (5 mM), respectively. The stabilization effect of 3-MA on NS5 (lane 4 in 3-MA-treated cells compared to lane 3 in PBS-treated cells) and the NS5-mediated acetylation of MTs are shown. The α-tubulin protein was used as the loading control protein. A representative experiment of two is shown (see associated data in ). Histograms quantifying the amount of NS5-HA expressed, normalized to total α-tubulin, in both control (PBS)- and 3-MA-treated cells from the top Western blot experiments. The data are presented as the means ± S.E.M. of two independent experiments. In ( a ), the p values are *** p ≤ 0.001 and * p ≤ 0.1, and in ( b ), the p value is 0.15. The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: HEK-293T cells (cat. number 103, NIH AIDS Research and Reference Reagent Program) and Lucia luciferase reporter HEK-293 cells expressing human RIG-I, HEK-Lucia™ RIG-I, (hkl-hrigi, InvivoGen, San Diego, CA, USA) were grown at 37 °C in a humidified atmosphere with 5% CO 2 in DMEM (Lonza, Verviers, Belgium) supplemented with 10% foetal calf serum (FCS) (Lonza), 1% L-glutamine and 1% penicillin–streptomycin (Lonza, Basel, Switzerland).

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot, Comparison