Journal: Frontiers in Cell and Developmental Biology

Article Title: Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells

doi: 10.3389/fcell.2022.829404

Figure Lengend Snippet: MICA/B release is increased from senescent cells. (A) SH-SY5Y cells were treated with 2 μM MLN8237, 0.5 μM DOX, DMSO, or no treatment as a control, and after 72 h, cellular senescence was evaluated by SA-β-gal staining. Senescent cells are shown by blue staining (magnification: ×20). (B) Cell cycle progression was assessed by flow cytometry. (C) Four different neuroblastoma cell lines were treated with 2 μM MLN8237, 0.5 μM DOX, DMSO, or no treatment as a control, and after 72 h, (D) MICA and MICB concentrations in the supernatant were evaluated by ELISA. Data represent the mean ± SEM of three independent experiments. * p < 0.05.

Article Snippet: The MICA(1:100, EK0812-CAP, BOSTER) or MICB(1:100, EK0963-CAP, BOSTER) monoclonal antibody was then added and incubated for another 1 h at 37°C, followed by washing the cells three times with TBS, addition of the diluted peroxidase complex, and incubation at 37°C for 30 min. After washing five times, 3,3′,5,5′-tetramethylbenzidine luminescent substrate was added, and color development was performed for 30 min.

Techniques: Control, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells

doi: 10.3389/fcell.2022.829404

Figure Lengend Snippet: GI254023X combination chemotherapy reduces MICA/B secretion and enhances NK cell recognition. IMR-32 cells were induced to senescence by treatment with MLN8237 or DOX, followed by addition of the ADAM10 inhibitor GI254023X (5 µM) for 72 h. The expression of (A) MICA and (B) MICB in the supernatant was detected by ELISA. (C) Cell-surface expression of MICA/B was detected by flow cytometry following the described treatments. (D) Cells in the different treatment groups were used as target cells, and normal human peripheral blood NK cells were used as effector cells. Killing efficiency was detected by the calcein-AM method in co-cultures at effector:target cell ratios of 5:1,10:1, and 20:1 for 4 h. Data represent the mean ± SEM of three independent experiments. * p < 0.05.

Article Snippet: The MICA(1:100, EK0812-CAP, BOSTER) or MICB(1:100, EK0963-CAP, BOSTER) monoclonal antibody was then added and incubated for another 1 h at 37°C, followed by washing the cells three times with TBS, addition of the diluted peroxidase complex, and incubation at 37°C for 30 min. After washing five times, 3,3′,5,5′-tetramethylbenzidine luminescent substrate was added, and color development was performed for 30 min.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry