Journal: Frontiers in Immunology
Article Title: Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters
doi: 10.3389/fimmu.2018.00662
Figure Lengend Snippet: Plasma membrane profiling (PMP) by mass spectroscopy identifies ABCG2. (A) Table of top hits identified using PMP to compare HMB-PP treated and untreated HeLa-M cells. Several independent peptides of ATP-binding cassette (ABC) transporter ABCG2 were detected, with modest increase (log 2 2.69) by treatment. Other potential candidates by these criteria (C3orf52 and FAM129B) are listed and were less significant. (B) Dysregulation of a subset of ABC transporters, including ABCG2, in HeLa cells detected by RT-PCR. Template cDNA was prepared from total RNA from HeLa-L and HeLa-M cells left untreated or treated with γδ T cell supernatant for 24 h. RT-PCR products were analyzed by gel electrophoresis. (C) Variation in ABCG2 protein expression in HeLa cells by western blot. Top panel: HeLa-L and HeLa-M cells were left untreated or treated with MG132 (5 µM for 10 h) or HMB-PP (10 nM for 10 h). Cell lysates were prepared and analyzed using antibodies directed to ABCG2, NRF2, and calnexin (CNX), which acts as a loading control. Bottom panel: western blot analysis of shRNA ABCG2 cells confirms reduction in ABCG2 protein expression. (D) HeLa-M cells show ABCG2-dependent resistance to cytotoxic drug doxorubicin (DOX). Wild-type HeLa-M, HeLa-L, and shRNA ABCG2 HeLa-M cells were incubated (24 h) with DOX at the indicated concentrations. Cells were stained using propidium iodide (PI) (2.5 µg/ml for 5 min) as a marker of plasma membrane integrity. After washing, cells were analyzed by cytometry. Chart shows mean fluorescence intensity (MFI) of PI staining. (E) No effect on T cell activation using shRNA ABCG2 HeLa-M cells. The effect on the efficiency of Vγ9/Vδ2 T cell activation for a series of HeLa-M gene knockdown lines, including shRNA BTN3A1, ABCG2, RhoB, PLEC1, and TRIM21. Cells were pretreated with HMB-PP (10 nM for 6 h) before Vγ9/Vδ2 T cells from a single donor were applied. Coculture was allowed to proceed overnight when culture supernatants were analyzed by ELISA. Chart shows percent (%) change in detected IFN-γ relative to wild-type HeLa-M cells of duplicate determinations for each cell line.
Article Snippet: Killing assay was by propidium iodide (PI) dye exclusion in cells treated with doxorubicin DOX (Cell Signaling).
Techniques: Clinical Proteomics, Membrane, Mass Spectrometry, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Expressing, Western Blot, Control, shRNA, Incubation, Staining, Marker, Cytometry, Fluorescence, Activation Assay, Knockdown, Enzyme-linked Immunosorbent Assay
