Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Designation of the DNA vaccine.(a) B cell epitope scanning of human DKK1 was performed with the software DNASTAR 7.1. (b) The affinity of epitopes was measured by indirect ELISA. (c) The separated epitopes were immunized BALB/c mice and the immunogenicity of epitopes was measured by sandwich ELISA. (d) The separated epitopes were injected to BALB/c mice for seven days. TRAP staining was performed to identify the mature osteoclasts. Magnification: 200x; data are expressed as the mean ± SEM.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Software, Indirect ELISA, Immunopeptidomics, Sandwich ELISA, Injection, Staining

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Construction of the DNA vaccine. (a) The maps of recombinant DKK1 DNA vaccine. (b) The amino acid sequence of human DKK1. (c) The recombinant amino acid sequence of DKK1 DNA vaccine. Red line, 110–144aa; green line, 153–181aa; orange line, 182–216aa; blue line, 228–253aa; black line, signal peptide; box, PADRE.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Recombinant, Sequencing

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Expression and immunogenicity of the DNA vaccine. (a–c) Expression of the multiepitope protein in vitro and in vivo were determined in cell culture supernatants by ELISA, cell lysates by Western blotting, and the muscles of mice by immunohistochemical analysis. (d-e) The DNA vaccine induced a specific IgG antibody against human DKK1. The titer and the end-point titer of the specific antibody were tested by ELISA. Bars indicate 100 μ m. Data are expressed as the mean ± SEM, * P < 0.05, *** P < 0.001.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Expressing, Immunopeptidomics, In Vitro, In Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Muscles, Immunohistochemical staining

Journal: Frontiers in Immunology

Article Title: Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1

doi: 10.3389/fimmu.2024.1365894

Figure Lengend Snippet: PAI-1 induces the expression of PD-L1 in human tumor cells. (A) Representative flow cytometric profiles of PD-L1 expression and MFI in the human cell lines HEK293T-WT, HEK293T-PAI-1 KO, ES2-WT, ES2-PAI-1 KO, MOLM14, K562-WT cells, and K562-PAI-1 OE cells treated with PAI-1 OE supernatant overnight. The experiments were repeated five times. (B) Representative flow cytometric profiles of PD-L1 expression and MFI ( n = 5/group) in HEK293T, ES2, MOLM14, and K562 cells after incubated with 100 nM rPAI-1 with or without 100 μM PAI-1 inhibitor TM5614. (C) Relative expression of PD-L1 mRNA in HEK293T, ES2, MOLM14, and K562 cells ( n = 5/cell type) after incubation with 100 nM rPAI-1 with or without 100 μM of the PAI-1 inhibitor TM5614. (D) Pearson’s correlation analysis between MFI of PAI-1 and MFI of PD-L1 in murine tumor cell lines (B16F1, B16F10, 32Dp210-WT, and 32Dp210-PAI-1 OE) and human tumor cell lines (HEK293T-WT, HEK293T-PAI-1 OE, ES2-WT, ES2-PAI-1 OE, K562-WT, and K562-PAI-1 OE). r: Pearson’s correlation coefficient. Bars are expressed as means ± SD of three independent experiments. ** p < 0.01, NS, non-significant.

Article Snippet: B16 (murine melanoma; F1 (parental), F10 (metastatic)), HEK293T (human embryonic kidney), ES2 (human clear cell ovarian carcinoma), and MOLM14 (human acute myeloid leukemia) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1

doi: 10.3389/fimmu.2024.1365894

Figure Lengend Snippet: PAI-1 promotes the production of soluble PD-L1, whereas blockade of PAI-1 suppresses the expression of PD-L1 expression both in vitro and in vivo. (A) Production of soluble PD-L1 in culture supernatants from MC38, B16F10, ES2, MOLM14, and K562 cells ( n = 5/cell type) after 4 days of incubation with 100 nM rPAI-1 with or without 100 μM of the PAI-1 inhibitor TM5614. (B) Representative flow cytometry graphs of PD-L1 expression and MFI were obtained from 32Dp210 cells engrafted in the spleen ( n = 6) and ES2 cells ( n = 6) engrafted in subcutaneous tissue at 14 days post-inoculation. Bars are expressed as means ± SD of three independent experiments. ** p < 0.01.

Article Snippet: B16 (murine melanoma; F1 (parental), F10 (metastatic)), HEK293T (human embryonic kidney), ES2 (human clear cell ovarian carcinoma), and MOLM14 (human acute myeloid leukemia) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, In Vitro, In Vivo, Incubation, Flow Cytometry

Journal: Cancer Research

Article Title: Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

doi: 10.1158/0008-5472.can-07-6885

Figure Lengend Snippet: Figure 1. BER does not influence cellular response to CNDAC. A, primer excision of CNDACMP by APE1 in vitro. Top, 5¶-[32P]–labeled 24-mer primers with either dCMP or CNDACMP at its 3¶ end were separately annealed to a 40-mer template to generate a recessed substrate (substrate A) to evaluate excision with 10 units APE1 for 0, 5, 10, 20, and 40 min, respectively. The reaction products were analyzed by 15% polyacrylamide sequencing gel and visualized by phosphoimager. Lanes 1-5, dCMP-terminated 24-mer/40-mer; lanes 6-10, CNDACMP-terminated 24-mer/40-mer. Bottom, a substrate containing a nick in the target strand was prepared by annealing the 5¶-[32P]–labeled 24-mer primer with CNDACMP at its 3¶ terminus to the 40-mer template, as above, in the presence of either of two 16-mer primers complementary to the distal portion of the template. The 5¶ terminus of one 16-mer had a phosphate, modeling the h-elimination product (substrate C), whereas the other 16-mer had a hydroxyl, which would reflect a dephosphorylated h-elimination product (substrate B). These two substrates were compared for excision by 10 units of APE1 for 0, 5, 10, 20, and 40 min, respectively. Lanes 1-5, substrate B, CNDACMP nicked DNA substrates with 5¶-OH 16-mer; lanes 6-10, substrate C, CNDACMP nicked DNA substrates with 5¶-PO4 16-mer. Results are representative of three experiments. B, the cytotoxicity of CNDAC seems to be independent of the BER pathway. Top, AA8 (n) and XRCC1-deficient EM9 cells (E; 500 cells per 60-mm dish) were treated with various concentrations of MMS for 2 h, then washed into drug-free medium, and incubated for 6 d. AA8 cells in the continuing presence of an APE1 inhibitor CRT0044876 (200 Amol/L; 5) were treated similarly. Middle, AA8 (with or without CRT0044876) and EM9 cells were treated with various concentrations of CNDAC for 24 h, then washed into CNDAC-free medium, and incubated for 6 d. Symbols, as above. Bottom, 92TAg wild-type (n) and 88TAg pol h–deficient (E) MEF cells were treated with CNDAC, as above. Colonies were counted and normalized as the percentage of control. Points, mean of three independent experiments; bars, SEM.

Article Snippet: The following cells were purchased from the American Type Culture Collection: the Chinese hamster ovary (CHO) AA8 cell line and its derived mutants EM9 (XRCC1), UV41 (XPF), UV20 (ERCC1), UV135 (XPG), UV24 (XPB), and UV5 (XPD); mouse embryonic fibroblast (MEF) cell lines 92Tag (wild type), 88Tag (pol h / ), and 127 Tag (PMS2 / ) and the cervical cancer line HeLa CCL2.

Techniques: In Vitro, Labeling, Sequencing, Incubation, Control

Journal: Cancer Research

Article Title: Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

doi: 10.1158/0008-5472.can-07-6885

Figure Lengend Snippet: Figure 3. The effect of CNDAC on the cytotoxicity of NER-deficient CHO cells (A and B) and human cells (C and D). A, left, AA8 and NER mutant cell lines (500 cells/60-mm dish) were treated with various concentrations of CNDAC for 24 h, then washed into drug-free medium and incubated for 6 d. Colonies were counted and normalized as the percentage of controls. AA8, n; UV41, E; UV20, !; UV135, x. Right, AA8 (.) and UV41 (E) cells were incubated with 0.01 Amol/L [3H]CNDAC for 24 h and washed into drug-free medium. This drug concentration did not affect clonogenic survival. Aliquots were taken at the indicated times, and DNA was extracted. The rates of removal from DNA were determined by quantitating the amount of each labeled nucleoside that remained in DNA by liquid scintillation counting. DPM, disintegration per minute. B, left, a human XPF gene cDNA expression plasmid, pcDNA3-XPF, was stably transfected into the UV41 cells and the clonogenic assays were then performed. The vector pcDNA3 plasmid was also transfected as a control. AA8, n; UV41, E; UV41 transfected with pcDNA3- XPF, .; UV41 transfected with vector, !. Right, immunoblotting of the cell lysates prepared from AA8, UV41, pcDNA3 transfected (vector), and pcDNA3-XPF transfected (hXPF) cells. As a positive control, the cell lysates from the HeLa cells were used in the assay. C, pcDNA3-XPF was stably transfected into the human XPF fibroblast GM08437B cells and the clonogenic assays were then performed. Cells transfected with pcDNA3-XPF, .; cells transfected with vector, !. Right, protein expression level of hXPF. D, sensitivity of XPF knockdown cell line to CNDAC is independent of p53. left, XPF protein in HCT116 p53 wild-type and null cells was knocked down by transient siRNA silencing and then treated with CNDAC for 24 h. Cell survival was assessed in clonogenic cell growth assays. p53 wild-type cells transfected with control siRNA, n; p53 wild-type cells transfected with siRNA targeting XPF, E; p53 null cells transfected with control siRNA, ; p53 null cells transfected with siRNA targeting XPF, .. Points, mean of three independent experiments; bars, SEM. Right, Western blot analysis of total protein content from control cells, nontargeting control siRNA, and XPF siRNA knockdown cells.

Article Snippet: The following cells were purchased from the American Type Culture Collection: the Chinese hamster ovary (CHO) AA8 cell line and its derived mutants EM9 (XRCC1), UV41 (XPF), UV20 (ERCC1), UV135 (XPG), UV24 (XPB), and UV5 (XPD); mouse embryonic fibroblast (MEF) cell lines 92Tag (wild type), 88Tag (pol h / ), and 127 Tag (PMS2 / ) and the cervical cancer line HeLa CCL2.

Techniques: Mutagenesis, Incubation, Concentration Assay, Labeling, Expressing, Plasmid Preparation, Stable Transfection, Transfection, Control, Western Blot, Positive Control, Knockdown

Journal: Cancer Research

Article Title: Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

doi: 10.1158/0008-5472.can-07-6885

Figure Lengend Snippet: Figure 5. The cytotoxicity of CNDAC is independent of GG-NER but dependent on TC-NER. XPC (n) and XPCC (E) cells were examined for their UV (A) and CNDAC (B) sensitivity, respectively. XPC, human fibroblast cell line derived from a patient with XPC syndrome; XPCC, XPC cells repleted with a human XPC cDNA. Protein level is shown in inset. C, XPC protein level was knocked down by siRNA transfection in HCT116 cells and cell survival was examined using clonogenic assay. HCT116 transfected with siRNA targeting XPC gene (E) and transfected with nontargeting control siRNA (n). Protein level is shown in inset. D, clonogenicity of AA8 (n), UV61 (E), UV5 (5), and UV24 (.) cells. Points, mean of three independent experiments; bars, SEM.

Article Snippet: The following cells were purchased from the American Type Culture Collection: the Chinese hamster ovary (CHO) AA8 cell line and its derived mutants EM9 (XRCC1), UV41 (XPF), UV20 (ERCC1), UV135 (XPG), UV24 (XPB), and UV5 (XPD); mouse embryonic fibroblast (MEF) cell lines 92Tag (wild type), 88Tag (pol h / ), and 127 Tag (PMS2 / ) and the cervical cancer line HeLa CCL2.

Techniques: Derivative Assay, Transfection, Clonogenic Assay, Control

Journal: Cancer Research

Article Title: Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

doi: 10.1158/0008-5472.can-07-6885

Figure Lengend Snippet: Figure 4. Cytotoxicity of ara-C and L-OddC to XPF-deficient CHO cells. AA8 and UV41 cells were treated with various concentrations of ara-C (A) or

Article Snippet: The following cells were purchased from the American Type Culture Collection: the Chinese hamster ovary (CHO) AA8 cell line and its derived mutants EM9 (XRCC1), UV41 (XPF), UV20 (ERCC1), UV135 (XPG), UV24 (XPB), and UV5 (XPD); mouse embryonic fibroblast (MEF) cell lines 92Tag (wild type), 88Tag (pol h / ), and 127 Tag (PMS2 / ) and the cervical cancer line HeLa CCL2.

Techniques:

Journal: Cancer Research

Article Title: Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

doi: 10.1158/0008-5472.can-07-6885

Figure Lengend Snippet: Figure 6. A, this table summarizes the contribution of NER proteins to resistance of CNDAC and UV. Note that all mutants exhibited UV sensitivity but only CSB, XPB, XPF, and ERCC1 exhibited CNDAC sensitivity, relative to AA8 wild-type cells. B, this model summarizes the participation of NER components in processing DNA damage caused by CNDAC. Both GG and TC-NER are required in order to excise the damage caused by UV, but TC-NER is the only pathway in processing the damage induced by CNDAC incorporation. The damage generated following incorporation of CNDAC actually is a CNddC-terminated nick created through h-elimination. When this nick is encountered by RNA polymerase II during transcription, CSB will be recruited to define this damage and further recruit helicase XPB for unwinding DNA helix in a 3¶ to 5¶ polarity and allowing access of XPF/ERCC1 to incise from 5¶ to lesion. The XPD helicase and XPG endonuclease are not necessary due to the existence of the nick. This could explain the sensitivity of UV61 (CSB), UV24 (XPB), and UV41 (XPF) and lack of effect for UV5 (XPD) and UV135 (XPG) in response to CNDAC compared with AA8 (wild type). Numbers indicate fold of sensitive. CN, CNddC.

Article Snippet: The following cells were purchased from the American Type Culture Collection: the Chinese hamster ovary (CHO) AA8 cell line and its derived mutants EM9 (XRCC1), UV41 (XPF), UV20 (ERCC1), UV135 (XPG), UV24 (XPB), and UV5 (XPD); mouse embryonic fibroblast (MEF) cell lines 92Tag (wild type), 88Tag (pol h / ), and 127 Tag (PMS2 / ) and the cervical cancer line HeLa CCL2.

Techniques: Generated

Journal: Bioorganic chemistry

Article Title: 1,3,4-oxadiazole/chalcone hybrids: Design, synthesis, and inhibition of leukemia cell growth and EGFR, Src, IL-6 and STAT3 activities

doi: 10.1016/j.bioorg.2018.11.032

Figure Lengend Snippet: GI 50 and TGI of compound 8v against the panel of 58 cell lines representing 9 different cancers tested using NCI’s in vitro five dose anticancer assay.

Article Snippet: Compound 8v exhibited LC 50 with concentrations ranging from 5.28 to > 100 μM. table ft1 table-wrap mode="anchored" t5 caption a7 Panel/Cell Line GI 50 (μM) TGI (μM) Panel/Cell Line GI 50 (μM) TGI (μM) Leukemia Melanoma CCRF-CEM 1.84 4.56 LOX IMVI 0.32 1.28 HL-60(TB) 2.00 4.10 MALME-3M 1.68 3.45 MOLT-4 2.05 5.27 M14 1.60 3.30 RPMI-8226 1.73 4.31 MDA-MB-435 1.70 3.40 SR 1.86 4.01 SK-MEL-2 1.79 4.82 Non-Small Cell Lung Cancer SK-MEL-28 1.74 3.40 A549/ATCC 1.84 3.53 SK-MEL-5 2.14 5.24 EKVX 1.79 3.98 UACC-257 2.16 4.69 HOP-62 2.13 4.53 UACC-62 1.73 3.43 HOP-92 1.93 4.23 Ovarian Cancer NCI-H226 2.67 5.69 IGROV1 1.54 3.75 NCI-H23 1.88 3.97 OVCAR-3 1.78 3.38 NCI-H322M 1.59 2.98 OVCAR-4 2.00 4.62 NCI-H460 2.00 3.85 OVCAR-5 1.72 3.30 NCI-H522 1.55 3.21 OVCAR-8 2.32 5.73 Colon Cancer NCI/ADR-RES 2.42 7.03 COLO 205 1.94 3.84 SK-OV-3 2.42 5.68 HCC-2998 1.88 3.65 Renal Cancer HCT-116 0.50 1.94 786–0 1.70 3.36 HCT-15 1.63 3.22 A498 11.00 23.60 HT29 1.83 3.62 ACHN 1.72 3.22 KM12 1.77 3.40 RXF 393 1.74 3.54 SW-620 1.86 3.60 SN12C 1.83 3.57 CNS cancer TK-10 1.52 3.03 SF-268 1.79 3.96 UO-31 1.46 2.97 SF-295 1.77 3.35 Breast cancer SF-539 1.64 3.03 MCF7 1.27 3.68 SNB-19 1.78 3.56 MDA-MB231/ATCC 1.82 3.50 SNB-75 1.47 3.15 HS 578T 2.65 7.76 U251 1.68 3.18 BT-549 1.40 2.81 Prostate Cancer T-47D 2.05 5.08 PC-3 1.75 3.30 MDA-MB-468 2.10 5.02 DU-145 1.75 3.21 Open in a separate window GI 50 and TGI of compound 8v against the panel of 58 cell lines representing 9 different cancers tested using NCI’s in vitro five dose anticancer assay.

Techniques: In Vitro

Journal: bioRxiv

Article Title: Interleukin-1 is overexpressed in injured muscles following spinal cord injury and promotes neurogenic heterotopic ossification

doi: 10.1101/2021.10.19.464906

Figure Lengend Snippet: IL-1β is expressed in human NHO biopsies and stimulates calcium mineralization of NHO-derived FAPs in vitro . (A) Representative immunohistochemistry staining of serial sections of a human NHO biopsy (SCI patient) demonstrating the colocalization of the macrophage marker CD68 (top panel left) and IL-1β protein expression (top panel right). Black rectangle shows IL-1β expressing CD68 + macrophages in the fibrotic tissue surrounding NHO while red rectangle shows IL-1β expressing multinucleated CD68 + osteoclasts. Mouse IgG3 (bottom left panel) and Rabbit IgG (bottom right panel) confirm the specificity of CD68 and IL-1β antibodies. 20X magnification and scale bar = 50μm. (B) IL-1β concentration in plasma of healthy volunteers and NHO patients. Each dot represents a different individual. Bars are means ± SD. Statistical difference was calculated using two-sided non-parametric Mann-Whitney test, *** p<0.001. (C) Dose-response of recombinant human IL-1α (0.01; 1 and 100 ng/mL) and (D) IL-1β (0.01; 0.5 and 10 ng/mL) on calcium mineralization of PDGFRα + CD56 - FAPs derived from 5 different NHO biopsies / patients cultured in osteogenic medium. (E) Osteogenic differentiation assay of FAPs cultured with LPS-stimulated CD14 + blood monocyte-conditioned medium (CM + ) and incubated with anti-IL-1α, anti-IL-1β neutralizing antibodies or isotype control (100 ng/mL). (F) Osteogenic differentiation assay of FAPs derived from 6 different biopsies / patients cultured with CM + and incubated with 10, 100 or 500 ng/mL of recombinant human IL1RA. In (C-E), each dot represents a result from a different NHO biopsy / patient. Boxes extend from the 25th to 75th percentiles, middle line and whiskers represent respectively median and minimum and maximum value. Statistical differences were analyzed using non-parametric repeated measure Friedman test with Dunn’s correction for multiple comparisons, * p < 0.05; ** p < 0.01. (G) RUNX2 protein expression level of FAPs evaluated by Western blot following 7 days of osteogenic differentiation with IL-1β (10 ng/mL), IL-1α (100 ng/mL), NHOmac CM + , control isotype antibody, anti-IL-1α and anti-IL-1β antibody (100 ng/mL) or IL-1RA (500 ng/mL). Statistical differences were analyzed using non-parametric repeated measure Friedman test with Dunn’s correction for multiple comparisons, * p < 0.05; ** p < 0.01.

Article Snippet: Human MPCs were trypsinized and incubated 30 min with biotinylated anti-human PDGFRα (BAF322 R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% fetal bovine serum (FBS), 2mM EDTA or with control isotypes IgG1 PE (A07796, Beckman Coulter) and biotinylated goat IgG (BAF108, R&D Systems).

Techniques: Derivative Assay, In Vitro, Immunohistochemistry, Staining, Marker, Expressing, Concentration Assay, Clinical Proteomics, MANN-WHITNEY, Recombinant, Cell Culture, Differentiation Assay, Incubation, Control, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 is dynamically expressed in skeletal muscle tissue. (A) Total RNAs were extracted from E14.5 and E19.5 mouse hind limb muscles and further subjected to RT-PCR to detect the mRNA expression of Fzd9 (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). GAPDH expression was used as a loading control gene. The gel is representative of two experiments performed by triplicate. (B) Proteins from mouse hind limb skeletal muscles obtained at different developmental stages (E14.5, E16.5, and E19.5) were fractionated by SDS-PAGE and immunoblotted for Fzd9 and β-actin ( upper panel ). Fzd9 is expressed at the protein level in all the analyzed developmental stages since the expected 56kDa band is detected. A representative gel shows that Fzd9 band intensity displays a progressive decrease from E14.5 to E19.5. Data represent the mean ± SD of Fzd9/β-actin ratio from three experiments, normalized to E14.5 ( lower panel ) ( * p < 0.05, ** p < 0.01, ANOVA using Bonferroni's post-hoc analysis). (C) Whole-mounted diaphragms from E17.5 and P0 were stained with anti Fzd9 antibody (green) together with αBTX to reveal the postsynaptic densities (blue). Fzd9 is abundant in the synaptic domain of embryonic NMJs, where it displays a punctate expression pattern. The insets ( lower panels ) show that some αBTX-positive regions were not labeled with anti Fzd9 antibodies. Pictures are representative of at least three experiments performed by triplicate.

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Muscles, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Reverse Transcription, Control, SDS Page, Staining, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 impairs agrin-dependent AChR clustering in myotubes. (A) Fzd9 is expressed in the muscle cell line C2C12 throughout differentiation. C2C12 cells were cultured in vitro and differentiated for 0, 3, or 6 days (d0-d6). Total proteins were subjected to Western blot analyses. An expected 56 kDa band is gradually increased during C2C12 cells differentiation. α-tubulin expression was used as a loading control. (B) C2C12 myotubes differentiated for 6 days were analyzed by immunocytochemistry to detect Fzd9. Fzd9 is localized to the plasma membrane of the myotubes (green, upper panel ), similar to Glut1, which was used as a marker of plasma membrane (red, middle panel ). The merge image ( lower panel ) reveals the co-localization of Fzd9 and Glut1. (C) Differentiated C2C12 myotubes were subjected to a sequential fractionation procedure to isolate samples enriched in cytoplasm (cyt) or plasma membrane (mb) proteins. Western blot analyzes showed that α-tubulin is specifically detected in cytoplasmic fractions, whereas the vitamin C transporter SVCT2 was only present in membrane-enriched protein fractions ( left panel ). C2C12 myoblasts were transfected either with GFP or Fzd9 and differentiated. Sequential protein lysates were analyzed by Western blot. Both endogenous Fzd9 (GFP-transfected cells) or overexpressed Fzd9 (Fzd9-transfected cells) were found predominantly in the plasma membrane and were absent in the cytoplasm. As a loading control, β-actin is found only in the cytoplasm-enriched fraction ( right panel ). (D) Myoblasts transfected with plasmids coding for GFP (control) or Fzd9 were differentiated into myotubes and subsequently incubated with 200 pM neural agrin. αBTX staining allows the visualization of the AChRs (red). Automatized quantification of aggregates shows that Fzd9 overexpression induces a decrease in the number of AChR clusters per myotube, as well as a reduction in the total area and average size of AChR clusters, compared to controls. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( ** p < 0.01, *** p < 0.001 compared to GFP controls, t -test).

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Cell Culture, In Vitro, Western Blot, Expressing, Control, Immunocytochemistry, Clinical Proteomics, Membrane, Marker, Fractionation, Transfection, Incubation, Staining, Over Expression

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Down-regulation of Fzd9 increases agrin-dependent AChR clustering in myotubes. (A,B) The efficiency of shFzd9 was tested by its ability to impair overexpression/function of the Fzd9 construct. (B) HEK293 cells were transfected with Fzd9HA together with a control shRNA (pFUX) or shFzd9, followed by protein homogenization and Western blot. Whereas a 56 kDa band corresponding to Fzd9 is detected in the membrane-enriched fraction of the control condition, Fzd9 expression is drastically silenced in cells transfected with shFzd9. (B) The efficiency of the shFzd9 to affect the functionality of Fzd9HA was assessed by co-transfecting Fzd9HA and Wnt2 in the presence or absence of the shFzd9 plasmid in HEK293 cells. Activation of the TOPflash luciferase reporter gene was used as a readout of activation of the canonical Wnt pathway. These experiments were performed at least three times by triplicate ( ** p < 0.01, *** p < 0.001, t -test). (C) Myoblasts were transfected either with GFP or shFzd9 and differentiated for 5 days. Myotubes were treated with neural agrin and further stained with an anti-Fzd9 antibody (red), along with αBTX (blue) to detect AChR clusters and DAPI (yellow) to stain nuclei. shFz9-transfected myotubes display silenced Fzd9 expression and an apparent increase in the number of AChR clusters compared to GFP-expressing myotubes. (D) Myoblasts transfected either with GFP or shFzd9 and grown for 5 days were treated with neural agrin and further stained with αBTX to detect AChR clusters (red). Myotubes expressing the GFP protein present in the shFzd9 plasmid show a significant increase in the number of AChR clusters, an increase in the total area of AChR clusters, as well as on the average size of AChR clusters, when compared to control myotubes that only express GFP. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( * p < 0.05, *** p < 0.001 compared to GFP controls, t -test).

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Over Expression, Construct, Transfection, Control, shRNA, Homogenization, Western Blot, Membrane, Expressing, Plasmid Preparation, Activation Assay, Luciferase, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 enhances β-catenin accumulation in myotubes. (A) GFP and Fzd9-transfected myotubes were immunostained with an anti β-catenin antibody (red). Fluorescence intensity of transfected myotubes was quantified using Metamorph. Quantification of the data ( right panel ) shows that the expression of Fzd9 induces a significant ~2-fold accumulation of β-catenin in the sarcoplasma, compared to control GFP-expressing myotubes. (B) Total protein samples from GFP- and Fzd9-transfected myotubes were separated by SDS-PAGE and immunoblotted with Fzd9 and β-catenin antibodies. Quantification of the Fzd9 or β-catenin against β-actin band intensity ratios shows that Fzd9-overexpressing myotubes display a ~2-fold increase in β-catenin cytosolic levels, which is equivalent to the ~2-fold increase observed for Fzd9 levels, compared to control myotubes ( right panel ). Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to control GFP cells; *** p < 0.001, t -test, compared to the GFP group). (C) Whole-mounted diaphragms of E17.5 mice were immunostained to detect Fzd9 (green) and β-catenin (red). AChR aggregates were stained with αBTX (blue). β-catenin is associated to the sarcolemma of embryonic muscle fibers, including the membrane domains where AChR clusters and Fzd9 are localized. Pictures are representative of at least three experiments performed by triplicate.

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Transfection, Fluorescence, Expressing, Control, SDS Page, Staining, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Nuclear β-catenin in vessels of human astrocytoma does not correlate with WHO grade. (A) TCGA database analyses for Wnt1 , Wnt3a , Wnt5a , Wnt7a , Wnt7b , Dkk1 , and Dkk2 mRNA expression as log2-fold expression. Differences in mRNA expression in GBM compared with normal central nervous system tissue (dashed red line) are shown. (B and C) Paraffin sections of three normal human brains (B) and five different human astrocytoma WHO grades I–IV (C) stained for β-catenin and analyzed for its endothelial, nuclear localization. Insets show individual nuclei in higher magnification. Bars: (B) 14 µm; (C) 20 µm.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Wnt1 expression decreased subcutaneous GL261 tumor growth and increased animal survival. (A) Western blots showing Wnt1 and Dkk1 expression +/−DOX in GL261 cells. (B) sTOP-FLASH assay on human embryonic kidney (HEK293)/GL261 co-cultures without DOX. Monoculture of transfected HEK293 cells served as baseline. Wnt1 and Dkk1 cells were cultured without (−) or with (+) Wnt3aCM (one experiment in triplicate). (C) In vitro proliferation of the control-, Wnt1-, and Dkk1-GL261 line cultured +/−DOX. (D, left) Representative pictures of NUDE mice with subcutaneous tumors −DOX. (right) Tumor volumes ( n = 7/group) of the transplanted glioma cell lines −DOX (*, P < 0.05; **, P < 0.01). (E) H&E-stained paraffin sections revealed reduced necrotic areas for Dkk1-expressing tumors (N). (F, left) Pimonidazole (brown) immunohistochemistry staining revealed tumor hypoxia, hematoxylin counterstaining (blue). (right) Hypoxia in Dkk1 −D compared with the control −D (*, P < 0.05) and Wnt1 −D tumors (**, P < 0.01; n = 4 tumors/group, slices from the center of similar sized tumors). (G, left) Representative vessels, stained for nuclear β-catenin (brown) and hematoxylin (blue) of control −D , Wnt1 −D , and Dkk1 −D tumors. Arrowheads point to β-catenin + nuclei. (right) Quantification of β-catenin + nuclei ( n = 4 tumors/group, 20 vessels/tumor; ***, P < 0.001). Bars: (D) 1 cm; (E and F) 400 µm; (G) 35 µm. Error bars indicate SEM.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Expressing, Western Blot, Transfection, Cell Culture, In Vitro, Control, Staining, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor-derived Wnt1 reduced and normalized subcutaneous tumor vascularization, whereas Dkk1 caused the opposite effect. (A) Tumor growth in Wnt1 −D compared with Wnt1 +D condition (left; n = 12/group; **, P < 0.01; ***, P < 0.001) and mouse survival (right; n = 12/group). (B) Tumor volume for Dkk1 −D compared with Dkk1 +D condition (left; n = 12/group; *, P < 0.05; **, P < 0.01; ***, P < 0.001) and mouse survival of Dkk1 +/−D tumors (right; n = 12/group). (C, top) IF staining on subcutaneous Wnt1 tumors for CD31/PECAM-1, α-SMA, and TOPRO-3. (bottom left) Vessel density of subcutaneous Wnt1 tumors +/−DOX ( n = 5 tumors/group, 10 pictures/tumor; **, P < 0.01). (bottom right) Association of α-SMA + cells to ECs in Wnt1 +/−D tumors ( n = 5 tumors/group, 10 vessels/tumor; ***, P < 0.001). (D) Same staining and experimental settings as in C for subcutaneous Dkk1 +/−D tumors (*, P < 0.05). (C and D) Insets show vessels in higher magnification. (E) Perfusion with isolectin revealed that vessels from control −D , Wnt1 −D , and Dkk1 −D tumors exhibited blood flow. Bars: (C and D) 400 µm; (E) 200 µm. Error bars indicate SEM.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Derivative Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Glioma-derived Wnt1 up-regulated endothelial Dll4 , leading to a stalk cell–like gene signature. (A) ISH against Dll4 (black) and hematoxylin (blue) of subcutaneous control −D , Wnt1 −D , and Dkk1 −D GL261 tumors, showing representative small (0–300 µm 2 ; left) and large (>300 µm 2 ; right) vessels. (B, left) Quantification of vessel diameter ( n = 3 tumors/group, 15 pictures/tumor) grouped in categories of 0–30 µm 2 , 31–300 µm 2 , and >300 µm 2 (*, P < 0.05; **, P < 0.01). (right) Quantification of Dll4 + ECs ( n = 6 tumors/group, 5 small and 5 large vessels/tumor; ***, P < 0.001). (C, top) HUVECs co-cultivated with control −D , Wnt1 −D , and Dkk1 −D GL261 cells (asterisks indicate HUVECs). (bottom) qRT-PCR ( n = 6) for human genes regulated by Wnt1 −D and Dkk1 −D compared with control −D co-cultures (red line). (D) qRT-PCR for mouse genes regulated in MBEs stimulated by Wnt3aCM for 18 and 48 h compared with controlCM (red line; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars: (A) 50 µm; (C) 80 µm. Error bars indicate SEM.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Derivative Assay, Control, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor cell–free Matrigel plug assay confirmed the antiangiogenic effect observed in Wnt1-GL261 tumors. (A) Immunohistochemistry staining for CD31/PECAM-1 (red) and hematoxylin counterstaining (blue) of Matrigel plug sections, supplemented with diluent (control), 400 µg/ml human Wnt1, or 200 µg/ml mouse Dkk1 recombinant proteins. Bar, 200 µm. (B) Quantification of CD31 + area in percentage in Matrigel plugs ( n = 6 plugs/group, 8 representative pictures/plug; **, P < 0.001). Error bars indicate SEM.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Matrigel Assay, Immunohistochemistry, Staining, Control, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor-derived Wnt1 normalized vascularization and retained vascular barrier properties in GL261 glioma. (A, left) Representative H&E staining of serial thick sections of tumor-bearing mouse brains. (right) Corresponding IF staining for Podxl and TOPRO-3. (B, top) Quantification of glioma volume based on H&E staining (control −D n = 3, Wnt1 −D n = 6, and Dkk1 −D n = 4; *, P < 0.05). (bottom) Podxl + vessel areas within tumors (control n = 4, Wnt1 n = 6, Dkk1 n = 5; **, P < 0.01). (C) IF staining for CD31/PECAM-1 and desmin ( n = 3 tumors/group, 8 pictures/tumor; *, P < 0.05). (D) Large image reconstructions of IF staining for mouse endogenous IgG (mIgG), Podxl, and TOPRO-3 on brain sections bearing control −D , Wnt1 −D , and Dkk1 −D glioma. Bars: (A) 200 µm; (C) 50 µm; (D, top) 1 mm; (D, bottom) 27 µm. Error bars indicate SEM.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Derivative Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Pdgfb is a Notch-independent target of β-catenin signaling in ECs. (A) HUVECs co-cultivated with control −D , Wnt1 −D , and Dkk1 −D GL261 cells. qRT-PCR ( n = 4) for human PDGFB regulated by Wnt1 −D or Dkk1 −D (ns) compared with control −D (gray line; *, P < 0.05). (B) qRT-PCR for Pdgfb gene regulated in MBEs stimulated by Wnt3aCM for 18 and 48 h compared with controlCM (gray line; n = 4; *, P < 0.05; ***, P < 0.001). (C, left) qRT-PCR for Axin2 and Pdgfb regulated in β-catenin–deficient ECs transduced with LefΔN-βCTA compared with the vector control (gray line; n = 4; *, P < 0.05; **, P < 0.01). (right) Representative Western blot ( n = 1) for PDGF-B from LefΔN-βCTA ECs compared with the vector control; top band shows the nonreduced (PDGF-BB) and bottom band reduced protein (PDGF-B). Densitometric analysis shows the summarized values for both forms of PDGF-B of samples loaded twice. (D) qRT-PCR of β-catenin–deficient ECs (control) or transduced with LefΔN-βCTA grown for 24 h either on gelatin- or Dll4-coated dishes ( n = 3). Notch pathway induction was monitored by Hes1 expression (*, P < 0.05; **, P < 0.01). Notch signaling was blocked by Dll4-Fc. Error bars indicate SEM.

Article Snippet: Polyclonal antibodies used were as follows: rabbit anti–β-galactosidase (MP Biomedicals), rabbit anti-Clnd3 (Invitrogen), rabbit anti-ColIV (AbD Serotec), rabbit anti–PDGF-B (AB Biotec), and goat anti–human Dkk1, goat anti–mouse Podxl, and goat anti–mouse Wnt1 (R&D Systems).

Techniques: Control, Quantitative RT-PCR, Transduction, Plasmid Preparation, Western Blot, Expressing

Journal: Foods

Article Title: Unripe Black Raspberry ( Rubus coreanus Miquel) Extract and Its Constitute, Ellagic Acid Induces T Cell Activation and Antitumor Immunity by Blocking PD-1/PD-L1 Interaction

doi: 10.3390/foods9111590

Figure Lengend Snippet: Effect of Rubus coreanus extract (RCE) on programmed cell death protein 1 (PD-1)/PD-1 ligand-1 (PD-L1) protein interaction using competitive enzyme-linked immunosorbent assay (ELISA) or cell-based assay. ( A , B ) The effect of RCE on the binding of PD-1 and PD-L1 in vitro using a competitive ELISA assay. ( C , D ) The effect of RCE on cell viability of PD-1/NFAT Jurkat cells ( C ) and PD-L1/aAPC CHO-K1 cells ( E ) using the Cell Counting Kit-8 (CCK) assay. Each of the cells were treated according to the indicated concentrations of RCE for 24 h. ( E ) The effect of RCE on cellular PD-1/PD-L1 blockade activity. PD-1/NFAT Jurkat effector cells and PD-L1/aAPC CHO-K1 target cells were co-cultured with RCE or αPD-L1 for 24 h at the indicated concentrations. The relative PD-1/PD-L1 blockade activity were measured by PD-1/NFAT luciferase reporter assay. ( F ) Effect of RCE on IL-2 cytokine release in co-cultured cell model. Cell culture media was analyzed to measure IL-2 level by cytokine ELISA. Data are presented as mean ± S.E. of three representative independent experiments. Asterisks indicate significant inhibition of PD-1/PD-L1 binding activity by each test inhibitor as compared with the vehicle-treated group; *** p < 0.001, ** p < 0.01, * p < 0.05, compared with the vehicle group.

Article Snippet: Jurkat cells expressing human PD-1 and NFAT reporter gene (PD-1/NFAT Jurkat cells) and CHO-K1 cells expressing human PD-L1 and a T cell receptor activator (PD-L1/aAPC CHO-K1 cells) were purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Cell Based Assay, Binding Assay, In Vitro, Cell Counting, Activity Assay, Cell Culture, Luciferase, Reporter Assay, Inhibition

Journal: Foods

Article Title: Unripe Black Raspberry ( Rubus coreanus Miquel) Extract and Its Constitute, Ellagic Acid Induces T Cell Activation and Antitumor Immunity by Blocking PD-1/PD-L1 Interaction

doi: 10.3390/foods9111590

Figure Lengend Snippet: Effect of ellagic acid on PD-1/PD-L1 interaction in vitro. ( A ) Effect of ellagic acid on PD-1/PD-L1 interaction was measured by competitive ELISA. Ellagic acid was treated as indicated concentration (0–120.9 μg/mL). ( B ) Ellagic acid binds to PD-1 and PD-L1. Pull-down assays were performed to detect the binding of ellagic acid with PD-1 or PD-L1. Binding by ellagic acid between human recombinant PD-1 or PD-L1 proteins were confirmed by Western blot using antibodies against PD-1 (upper panel) or PD-L1 (lower panel). Lane 1: Recombinant PD-1 or PD-L1 protein alone; lane 2: each of the proteins were precipitated with Sepharose 4B-alone; lane 3: each of the proteins were precipitated with ellagic acid–Sepharose 4B beads (EA-Sepharose 4B). ( C , D ) Effect of ellagic acid on cell viability of (c) PD-1/NFAT Jurkat cells or ( D ) PD-L1/aAPC PD-L1 CHO-K1 cells. The cell viabilities were analyzed using the Cell Counting Kit-8 (CCK) assay. Cells were treated with or ellagic acid as indicated concentrations (0–120.9 μg/mL). ( E ) Effect of ellagic acid on cellular PD-1/PD-L1 blockade activity. PD-1/NFAT Jurkat effector cells and PD-L1/aAPC CHO-K1 target cells were co-cultured with ellagic acid for 48 h at the indicated concentrations (0–7.56 μg/mL). The relative PD-1/PD-L1 blockade activity were measured by PD-1/NFAT luciferase reporter assay. ( F ) Effect of ellagic acid on IL-2 cytokine release in co-cultured cell model. Cell culture media was analyzed to measure IL-2 level by cytokine ELISA. Data are presented as mean ± S.E. of three representative independent experiments. Asterisks indicate significant inhibition of PD-1/PD-L1 binding activity by each test inhibitor as compared with the vehicle-treated group; *** p < 0.001, ** p < 0.01, * p < 0.05, compared with the vehicle group.

Article Snippet: Jurkat cells expressing human PD-1 and NFAT reporter gene (PD-1/NFAT Jurkat cells) and CHO-K1 cells expressing human PD-L1 and a T cell receptor activator (PD-L1/aAPC CHO-K1 cells) were purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: In Vitro, Competitive ELISA, Concentration Assay, Binding Assay, Recombinant, Western Blot, Cell Counting, Activity Assay, Cell Culture, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay, Inhibition