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 4. TGF-β treatment increased HK-2 cells susceptibility to NK-mediated lysis via NKG2D mediated pathway. (A) PBL cytotoxicity induced by treat ment of HK-2 cells with TGF-β (500 pg/ml) for 48 h. HK-2 cells were incubated with or without TGF-β (500 pg/ml) for 48 h. Target cells were subsequently harvested, labeled with calcein-AM for 10 min, washed with phosphate-buffered saline, and loaded at an effector to target cell ratio of 50:1 (black-filled bars), 25:1 (gray-filed bars), and 10:1 (open bars). After a 4-h incubation period, the specific lysis activity of the PBL was analyzed using a fluorescence reader. (B) Cytotoxicity of PBL against HK-2 [treated with TGF-β (500 pg/ml)] was assessed after pre-incubating PBL (5x106 cells/well) with IL-2 (500 U/ml) for 24 h. PBL cytotoxicity was induced by treatment of HK-2 cells with TGF-β (500 pg/ml) for 48 h. Target HK-2 cells were incubated with TGF-β (500 pg/ml) for 48 h. Target cells were harvested, labeled, washed and loaded at an effector to target cell ratio of 50:1, as described above. PBL and target cell mixtures were subsequently cultured with the indicated antibodies (1 µg/ml) for an additional 4 h. (C) HK-2 cells were treated with or without TGF-β (500 pg/ml) for 48 h. The MICA secretion level in culture supernatants was analyzed by an enzyme-linked immunosorbent assay. (D) Cytotoxicity of NK92-MI cells against HK-2 [treated with or without TGF-β (500 pg/ml)] was assessed after pre-incubating NK92MI cells (2x106 cells/well) with complete media (untreated) or with TGF-β (500 pg/ml) plus HK-2 cells (2x106 cells/well) for 48 h. TGF-β treated and untreated HK-2 cells and NK cells were co-cultured using a Transwell system. The HK-2 cells treated or untreated with TGF-β for 48 h were used as the target cells. Results are representative of three independent experiments. *P<0.05 vs. control. TGF, transforming growth factor; NKG2D, NK group 2 member D; PBL, peripheral blood lymphocytes; IL, interleukin; MICA, major histocompatibility complex class I-related chain molecules A.
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: Lysis, Incubation, Labeling, Saline, Activity Assay, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Immunopeptidomics
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![Figure 4. TGF-β treatment increased HK-2 cells susceptibility to NK-mediated lysis via NKG2D mediated pathway. (A) PBL cytotoxicity induced by treat ment of HK-2 cells with TGF-β (500 pg/ml) for 48 h. HK-2 cells were incubated with or without TGF-β (500 pg/ml) for 48 h. Target cells were subsequently harvested, labeled with calcein-AM for 10 min, washed with phosphate-buffered saline, and loaded at an effector to target cell ratio of 50:1 (black-filled bars), 25:1 (gray-filed bars), and 10:1 (open bars). After a 4-h incubation period, the specific lysis activity of the PBL was analyzed using a fluorescence reader. (B) Cytotoxicity of PBL against HK-2 [treated with TGF-β (500 pg/ml)] was assessed after pre-incubating PBL (5x106 cells/well) with IL-2 (500 U/ml) for 24 h. PBL cytotoxicity was induced by treatment of HK-2 cells with TGF-β (500 pg/ml) for 48 h. Target HK-2 cells were incubated with TGF-β (500 pg/ml) for 48 h. Target cells were harvested, labeled, washed and loaded at an effector to target cell ratio of 50:1, as described above. PBL and target cell mixtures were subsequently cultured with the indicated antibodies (1 µg/ml) for an additional 4 h. (C) HK-2 cells were treated with or without TGF-β (500 pg/ml) for 48 h. The MICA secretion level in culture supernatants was analyzed by an enzyme-linked immunosorbent assay. (D) Cytotoxicity of NK92-MI cells against HK-2 [treated with or without TGF-β (500 pg/ml)] was assessed after pre-incubating NK92MI cells (2x106 cells/well) with complete media (untreated) or with TGF-β (500 pg/ml) plus HK-2 cells (2x106 cells/well) for 48 h. TGF-β treated and untreated HK-2 cells and NK cells were co-cultured using a Transwell system. The HK-2 cells treated or untreated with TGF-β for 48 h were used as the target cells. Results are representative of three independent experiments. *P<0.05 vs. control. TGF, transforming growth factor; NKG2D, NK group 2 member D; PBL, peripheral blood lymphocytes; IL, interleukin; MICA, major histocompatibility complex class I-related chain molecules A.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1146/pm26311146/pm26311146__page5_image2.jpg)