Recombinant il 10

A549 cells are an alveolar type-II epithelial cell line, that were cultured in DMEM (Gibco, CA, USA), supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10 % fetal bovine serum (FBS) (Gibco, CA) at 37 °C, 5 % CO₂. Cells were seeded in 96-well tissue culture plates, following 60–70 % adherence. 100 ng/mL recombinant IL-17A (rOPN, R&D, MN, USA) and 15 μg/mL pulmonary Mycobacterium bovis bacille Calmette–Guerin (BCG, Shanghai Institute of Biological Products Co., Ltd, China) were added respectively. Cells and supernatants were harvested at 24 h. The supernatants were used to determine the concentration of lactate dehydrogenase (LDH) according to the manufacturer’s instructions (Roche, Mannheim, Germany). An analysis of cellular apoptosis was carried out according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA, USA).
PBMCs from health volunteers were cultured at 37 °C, 5 % CO₂ in RPMI 1640 (Gibco, CA, USA), supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10 % FBS. Cells were incubated with recombinant IL-10 (10 μg/mL, Biolegend, CA, USA) and BCG (15 μg/mL). Golgi inhibitor was added for the last 5 h of the incubation. Cells were harvested at 24 h, and intracellular cytokine analysis of IL-17A production was conducted according to the manufacturer’s instructions.

All flow cytometry sorting experiments were performed using a BD FACSAria cell sorter and analyzed by BD FACSDIVA™ software. Patients primary ATL cells, HuT-102 or CEM cell lines were stained with anti CD25-FITC (BD Biosciences, 2 ug/ml; US) before sorting. Sorted cells were analyzed by real-time PCR for Tax and HBZ expression. When indicated, sorted GFP-positive cells were treated with recombinant IL-10 (1 ng/ml) (Biolegend, #571006, US) and cell proliferation was assessed.
Twenty-four hours following transduction of cell lines (HuT-102, MT-1) or primary ATL cells with GFP-lentiviral vectors encoding scrambled (SCR), shRNA Tax or shRNA HBZ, cells were sorted based on GFP expression. The proliferation of GFP⁺ or GFP⁻ cells was assessed using the trypan blue dye exclusion assay. The expression of Tax, HBZ and IL-10 transcripts was assessed by real-time PCR. The expression of Tax protein was assessed by western blot. Cellular localization of RelA (Cell signaling; #D14E12; USA) and PML (homemade, gift from H. de Thé) in these cells was assessed by immunofluorescence assay and confocal microscopy analysis. Finally, protein expression of Dec-1 (Novus biologicals, #NB100-10200; USA), PML (homemade, gift from H.de The), p53 (Santa cruz; #DO-1; Germany), P-p53 (cell signaling #9284; USA) and P-IκBα (Invitrogen, #MA5-15224 US) was assessed by western blot.