Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Effect of Klotho on cellular viability in HUVECs. Cells were exposed to various concentrations of ox-LDL (25, 50, 100, and 200 μg/ml) for 24 h. a HUVEC morphology was observed under an inverted phase contrast microscope (×10) following 24 h of ox-LDL treatment. Typical cellular fragmentations, vacuoles, and debris are arrowed. (Bar = 50 μm) ( b ) Cellular viability was detected by MTT assay. c and d SOD enzymatic activity and MDA levels in HUVECs were analyzed using commercially available assay kits. e HUVECs were pretreated with 100, 200, 400, and 800 pM of Klotho for 1 h, and then incubated with ox-LDL (50 μg/ml) for 24 h. Cellular viability was detected by MTT assay. Data are shown as mean ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Microscopy, MTT Assay, Activity Assay, Incubation, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho inhibited ROS production induced by ox-LDL in HUVECs. HUVECs were pre-incubated with 200 pM of recombinant human Klotho for 1 h, then treated with ox-LDL (50 μg/mL) for another 24 h. Ox-LDL alone and Klotho alone were used as controls. a Images observed under an inverted fluorescence microscope. (Bar = 50 μm) ( b ) Output figure of the fluorescence intensity detected by flow cytometry. c Average fluorescence intensity: Mean = total area under the peak/the total number of cells. Data are shown as mean ± S.D. d Lipid peroxidation was assessed by measuring the MDA levels in HUVECs treated with ox-LDL and/or Klotho ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. blank control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Incubation, Recombinant, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: SOD, Cu/Zn-SODandgp91 phox in ox-LDL and Klotho-treated HUVECs. Cellular treatment was the same as described in Fig. . a Intracellular SOD activity was determined by the hydroxylamine method. b , c and d Western blotanalysis of Cu/Zn-SOD and gp91 phoxexpression in pre-treated HUVECs. Densitometry of the probed bands from the western blot were analyzed by Image J2x. Values are means ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Activity Assay, Western Blot, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho regulated NO production in HUVECs. a NO production in pre-treated HUVECs. b , c , d , and e mRNA levels of iNOS, eNOS, PI3K, and Akt were measured by real time PCR. Values are means ± S.D. ( n = 3). Statistical differences are expressed as # p < 0.05 vs . control; * p < 0.05 vs . ox-LDL. ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Tumor-associated antigen expression (normalized MFI, mean fluorescence intensity) in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Expressing, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Structure and in vitro activity of PDAC-directed T cell-engaging bispecific antibodies ( A ) IgG-[L]-scFv structure of BsAbs. CH, constant heavy chain; CL, constant light chain; scFv, single chain variable fragment; VH, variable heavy chain; VL, variabl light chain. ( B ) Flow cytometric analysis displaying fluorescent intensities of IgG-[L]-scFv BsAbs bound to human PDAC cells. IgG-[L]-scFv BsAbs evaluated were specific for human EGFR, HER2, MSLN, c-MET, and B7-H3. ( C ) Antibody-dependent T cell-mediated cytotoxicity (ADTC) as a function of increasing doses of BsAbs against PDAC cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Activity Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: In vitro sensitivities (EC50, pM) to target antigen-specific bispecific antibodies in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFR and HER2 T-BsAb heterodimerization and in vitro kinetics ( A ) Schematic of BsAb heterodimerization by controlled Fab Arm Exchange. Parental BsAbs bore 2 Fabs specific to EGFR or HER2 and 2 scFvs specific for CD3 (‘2 + 2’). Reduction of inter-H-chain disulfide bonds and subsequent heterodimerization (driven by F405L and K409R amino acid substitutions) yielded EGFRxHER2 BsAbs bearing 1 EGFR Fab, 1 HER2 Fab, and 2 CD3 scFv (‘1 + 1 + 2’). ( B ) SPR analysis of T-BsAbs binding to EGFR (left panel), HER2 (middle panel), and EGFR x HER2 (right panel) proteins. Representative normalized sensorgrams at 20nM were shown. ( C ) IHC staining of two cell-derived PDAC xenograft tumors SW1990 (top) and BxPC-3 (bottom). OCT-embedded tumor sections were stained with EGFRxEGFR, HER2xHER2, or EGFRxHER2 BsAbs. Control slides were either unstained (SW1990) or incubated with a control antibody (BxPC-3). ( D ) Antibody-dependent T cell-mediated cytotoxicity assays (ADTC) against SW1990 (left) and BxPC-3 (right). Cytotoxicity measured by Chromium-51 release. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. EC50s (SW1990; BxPC-3): EGFRxEGFR: 165.8fM; 31.5fM, HER2xHER2: 18.5pM; 7.14pM, EGFRxHER2: 699.6fM; 128.8fM. CD33xCD33 BsAb was included as a negative control

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Binding Assay, Immunohistochemistry, Derivative Assay, Staining, Control, Incubation, Negative Control

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Avidities of EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Protein Binding

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Heterodimeric EGFR and HER2 T-BsAbs impede PDAC growth ( A ) In vivo antitumor effect of BsAbs in the presence of human T cells against SW1990 cell line xenografts; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. T-BsAbs were administered twice per week and 2 × 10 7 of luciferase-transduced T cells (Luc(+) T cells) were administered once per week for 2 weeks to treat the tumors. ( B ) In vivo anti-tumor effects of 10 µg EGFR and HER2 T-BsAbs. ( C ) Relative body weight of mice during treatment and ( D ) overall survival were plotted. ( E ) Bioluminescence imaging (BLI) of Luc(+) T cells. Quantitation of bioluminescence intensity in the lesions of tumors. Representative images (right) were taken on day 7

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: T-BsAbs drive T cell infiltration, activation, and function in PDAC ( A ) Treatment of SW1990 tumor-bearing mice with EGFR and HER2 T-BsAbs; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. Once tumor reached ∼500mm 3 , mice received a single infusion of 2 × 10 7 T cells and were treated with 10µg T-BsAb, administered twice per week. Tumors were harvested for analysis on day 10 after initiation of treatment. Mice that exhibited 50% or more reduction in tumor volume by day 10 were excluded from this and subsequent analyses. ( B ) Flow cytometric analysis of the in vivo effect of T-BsAb treatments on T cell infiltration into harvested SW1990 PDAC tumors. Infiltrating T cells were identified by detection of human CD45 + and human CD4 + or CD8 + . ( C ) Frequency of T cell activation indicated by detection of IFN-γ + TNF-α + T cells and expression of CD69 among CD4 + or CD8 + T cells. ( D ) Frequency of CD11b + intratumoral myeloid cells and prevalence of PDL1 expression

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Activation Assay, In Vivo, Expressing

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: ADTC sensitivities (EC50, pM) of SW1990 lines to EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques:

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: FACS binding (EC50, pM) of EGFR and HER2 T-BsAbs to SW1990 lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFRxHER2 T-BsAbs efficacy is lost in EGFR and HER2-single-positive PDAC ( A ) Mean fluorescent intensities determined by flow cytometry and ( B ) antibody-dependent T cell-mediated cytotoxicity (ADTC) against SW1990-EGFR-KO (left) and SW1990-HER2-KO (right) cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. ( C ) Tumor growth of subcutaneous SW1990 EGFR-KO (left) and HER2-KO (right) tumors. One infusion of 2 × 10 7 T cells was administered. Two doses of T-BsAbs (10 µg each) were given by i.v. injection

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Flow Cytometry, Injection