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

Journal: Frontiers in Immunology

Article Title: Functional Characterisation and Analysis of the Soluble NKG2D Ligand Repertoire Detected in Umbilical Cord Blood Plasma

doi: 10.3389/fimmu.2018.01282

Figure Lengend Snippet: Soluble NKG2D ligand (sNKG2DL) effect on NK group 2, member D (NKG2D) and CD107a expression by CD56 bright CD3 − , CD56 dim CD3 − natural killer (NK) cells, CD56 + CD3 + NKT cells, CD56 − CD3 + T cells, and IFN-γ production by peripheral blood mononuclear cells (PBMCs). (A) Correlation and linear regression analysis of sULBP1 concentration effect on CD107a expression. (B) Correlation and linear regression analysis of sULBP1 and sMICB concentration on NKG2D expression. (C) Correlation and linear regression analysis of sULBP1 effect on production of IFN-γ by PBMCs. PBMCs were incubated with 50% cord blood plasma (CBP) ( n = 181) or media only for 48 h and then stimulated with PMA and ionomycin. IFN-γ in culture supernatants and sNKG2DLs in CBP was measured by ELISA and expression of CD107a and NKG2D on the different cell types was measured by flow cytometry. P -values for Spearman r correlations are indicated. (D) Increasing concentration of soluble ULBP1 decreases potential IFN-γ production by NK cells in a dose-dependent manner ( n = 181). Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis (D) was performed using Mann–Whitney test ± SEM (* P ≤ 0.05 and *** P < 0.001).

Article Snippet: Soluble MICA/B (DY1300/DY1599) and ULBP1 (DY1380) were detected in CBP and IFN-γ (DY285) was detected in PBMC stimulation supernatants using Duoset ELISA kits (R&D Systems, Abingdon, UK), according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay, Incubation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY