Journal: Experimental and therapeutic medicine

Article Title: Upregulated unique long 16 binding protein 1 detected in preeclamptic placenta affects human extravillous trophoblast cell line (HTR-8/SVneo) invasion by modulating the function of uterine natural killer cells.

doi: 10.3892/etm.2017.4143

Figure Lengend Snippet: Figure 2. Expression levels of ULBP1 in placentas from pregnant women with PE and women with NP were determined by RT‑qPCR, western blotting, and immunohistochemistry analysis. (A) RT‑qPCR analysis of ULBP1 mRNA expression levels in placentas from PE and NP women (n=30 for each group). (B) Western blot analysis of ULBP1 protein expression in placentas from PE and NP women. Upper panel, a typical result of western blotting; lower panel, bar chart according to the statistical analysis based on the result of three independently repeated experiments (n=30 for each group). (C) Immunostaining of ULBP1 in placentas from PE and NP women. Data are presented as the mean + standard error of the mean. Left panel, PE; middle panel, NP; right panel, negative control; scale bars, 200 µm. *P<0.05, PE vs. NP. ULBP1, unique long 16 binding protein 1; PE, preeclampsia; NP, normal pregnancy; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: Membranes were blocked for 1 h at room temperature with TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.0) containing 5% nonfat dry milk and incubated with primary antibodies against ULBP1 (1:200; sc-33456; Santa Cruz Biotechnology) and β-actin (1:8,000; 66009-1; Proteintech Group, Inc., Chicago, IL, USA) overnight at 4 ̊C.

Techniques: Expressing, Western Blot, Immunohistochemistry, Immunostaining, Negative Control, Binding Assay, Polymerase Chain Reaction

Journal: Experimental and therapeutic medicine

Article Title: Upregulated unique long 16 binding protein 1 detected in preeclamptic placenta affects human extravillous trophoblast cell line (HTR-8/SVneo) invasion by modulating the function of uterine natural killer cells.

doi: 10.3892/etm.2017.4143

Figure Lengend Snippet: Figure 3. uNK cell supernatants cultured with ULBP1 inhibited HTR‑8/SVneo cell invasion. (A) Representative images of invaded cells cultured with different CM in the Transwell invasion assay. Upper panel, the CM is uNK cell supernatants cultured with ULBP1; lower panel, the CM is uNK cell supernatants cultured without ULBP1; scale bars, 200 µm. Cells were stained with hematoxylin and eosin (magnification, x100). (B) Statistical bar graphs exhibiting the effect of uNK cell supernatants cultured with or without ULBP1 on the invasion of HTR‑8/SVneo cells in a Transwell invasion assay. Data are expressed as invasion index (n=10 in duplicate). (C) Statistical bar graphs exhibiting the effect of the addition of IFN‑γ neutralizing Ab to the uNK cell supernatants cultured with ULBP1 on invasion of HTR‑8/SVneo cells in a Transwell invasion assay (n=10 in duplicate). (D) Statistical bar graphs exhibiting the effect of the addition of IL‑8 neutralizing Ab to the uNK cell supernatants cultured with ULBP1 on invasion of HTR‑8/SVneo cells in a Transwell invasion assay (n=10 in duplicate). Data are presented as the mean + standard error of the mean. *P<0.05. uNK, uterine natural killer; ULBP1, unique long 16 binding protein 1; HTR‑8/SVneo, extravillous trophoblast cell line; IFN, interferon; Ab, antibody; IL, interleukin; CM, condition medium.

Article Snippet: Membranes were blocked for 1 h at room temperature with TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.0) containing 5% nonfat dry milk and incubated with primary antibodies against ULBP1 (1:200; sc-33456; Santa Cruz Biotechnology) and β-actin (1:8,000; 66009-1; Proteintech Group, Inc., Chicago, IL, USA) overnight at 4 ̊C.

Techniques: Cell Culture, Transwell Invasion Assay, Staining, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CHMP2A regulates broad immune cell-mediated antitumor activity in an immunocompetent in vivo head and neck squamous cell carcinoma model

doi: 10.1136/jitc-2023-007187

Figure Lengend Snippet: EVs secreted by 4MOSC cell lines express mouse NKG2D ligands. (A) 4MOSC1 and 4MOSC2 WT and mCHMP2A KO derived EVs were collected and analyzed by flow cytometry for mouse NKG2D ligands mRae, mULBP, mH60 (blue histogram). Red histogram represents isotype. EVs, extracellular vehicles; KO, knock-out.

Article Snippet: Isolated EVs were incubated with primary staining with mRae (AF1136-SP, R&D Systems) mULBP (MAB2588-SP, R&D Systems), and mH60 (MAB1155-SP, R&D Systems) for 30 min on ice and washed with flow buffer.

Techniques: Derivative Assay, Flow Cytometry, Knock-Out

Journal: International journal of molecular medicine

Article Title: Transforming growth factor-β1 regulates human renal proximal tubular epithelial cell susceptibility to natural killer cells via modulation of the NKG2D ligands.

doi: 10.3892/ijmm.2015.2317

Figure Lengend Snippet: Figure 1. Expression of NKG2D ligands by renal proximal tubular epithelial cell. (A) Surface and intracellular staining of NKG2D ligand proteins. Human renal proximal TECs (HK-2) were stained with control normal mouse immunoglobulin G (iso) or anti-MICA, and anti-ULBP1, 2 or 3 antibodies, and analyzed by flow cytometry. In the histograms, the gray peak represents the unstained control. (B) TGF-β-induced expression of NKG2D ligands. Renal proximal TECs (HK-2) were incubated in the presence of TGF-β (500 pg/ml) for 48 h, and surface and intracellular expression of NKG2D ligands were analyzed by flow cytometry. Results are representative of three independent experiments. NKG2D, NK group 2 member D; TECs, tubular epithelial cells; MICA, major histocompatibility complex class I-related chain molecules A; ULBP, UL16‑binding proteins; TGF, transforming growth factor.

Article Snippet: Cells were washed twice with ice-cold phosphate-buffered saline (PBS), and incubated with mouse anti-MICA antibody (#MAB13001) or anti-human ULBP monoclonal antibodies [anti-ULBP1 (#MAB1380), anti-ULBP2 (#MAB1298) and anti-ULBP3 (#MAB1517); R&D Systems} for 30 min on ice.

Techniques: Expressing, Staining, Control, Flow Cytometry, Incubation, Immunopeptidomics

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: List of primers used in qRT-PCR.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques:

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: Gemcitabine up-regulates NKG2D ligands in lung cancer cells. (A) qRT-PCR of H60, Raet-1 , and Ulbp1 mRNA in LLC cells. (B) qRT-PCR of major histocompatibility complex class I polypeptide-related sequence A ( MICA ), MICB , and UL16 binding protein ( ULBP ) 1-6 mRNA in A549 cells. Data are the fold-change of mRNA expression in gemcitabine- and cisplatin-treated cells relative to untreated cells. (C) Surface expression of MICA/B, ULBP1, ULBP2/5/6, and ULBP3 was measured by flow cytometry on A549 cells treated with vehicle control (DMSO), gemcitabine or cisplatin. Data are representative of three independent experiments. One-way analysis of variance (ANOVA) was used. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Immunopeptidomics, Sequencing, Binding Assay, Expressing, Flow Cytometry, Control