N acetylcystein

Organoids were established as described previously (15 (link)). In brief, cells were counted under a microscope and centrifuged at 500 × g for 5 minutes. Then, cells were resuspended in ice-cold 500 μL Matrigel (Corning) and 20 μL drops of Matrigel cell suspension were seeded on prewarmed 48-well culture plates (Corning) at a density of ∼2 × 10³ cells per 20 μL Matrigel/well. The Matrigel was solidified for 15 minutes at 37°C and overlaid with 250 μL airway organoid medium [AO; AdDF+++, 20% conditioned R-spondin1 medium supplemented with B27 (Invitrogen), 1.25 mmol/L N-acetylcystein (Sigma-Aldrich), 5 mmol/L nicotinamide (Sigma), 25 ng/mL human fibroblast growth factor 7 (Peprotech), 100 ng/mL human noggin (Peprotech), 100 ng/mL human FGF 10 (Peprotech), 500 nmol/L A83–01 (Tocris), and 500 nmol/L SB202190 (Sigma)]. AdDF+++ medium is advanced DMEM/F12 medium (Invitrogen) supplemented with 10 nmol/L HEPES (Invitrogen), 1× GlutaMax (Invitrogen), and 1× antibiotic–antimycotic (Invitrogen). Y-27632 (10 μmol/L; Enzo Life Science) was added for the first 2 days. Cultures were kept at 37°C, 5% CO₂ in a humidified incubator. Medium was replenished every 2 to 3 days.

Folin-Ciocalteu reagent, gallic acid, quercetin, DPPH, ABTS, 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ), ferrozine [3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate], trizma base, trichloroacetic acid, and ascorbic acid were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), fetal bovine serum (FBS), penicillin-streptomycin (P/S), phosphate-buffered saline (PBS), and trypsin-ethylenedi-aminetetraacetic acid (EDTA) were purchased from Gibco (Gaithersburg, MD, USA). Dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), insulin, Oil red O, nitroblue tetrazolium (NBT), and N-acetyl cystein were also purchased from Sigma-Aldrich Co.. All other chemicals were of reagent grade.

B-cells were plated at 10⁶ per ml and treated with purified recombinant Tat protein produced by Ablinc and obtained through the NIH AIDS Research and Reagent Program. According to the manufacturer, the Tat protein was >95% pure and purified by heparin-affinity chromatography and reverse phase HPLC which removed endotoxins [59] (link). If not stated differently, Tat was used for all experiments at a concentration of 250 ng/ml. TatC22 is a mutant Tat protein with a 22 Cysteine → 22 Glycine substitution whose transactivation function was abrogated [60] (link), [61] (link). Produced by Abcam, the TatC22 recombinant protein was used at 250 ng/ml. Some Tat treatments of B-cells were performed in the presence of the ROS scavenger Tempol at a final concentration of 10 μM (Sigma Aldrich) or N-acetylcystein at 1 mM (NAC; Sigma Aldrich) or the mitochondrial complex I inhibitor Rotenone at 10 μM (Sigma Aldrich) or a specific mitochondria-targeting antioxidant 10-(6’-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1) at 20 ng/ml (synthesized by G.A. Korshunova and N.V. Sumbatyan, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University) or the mitochondrial uncoupler 2,4-Dinitrophenol (DNP) at 10 µM, or Bay 11-7082, a NF-κB inhibitor, at 1 mM (Calbiochem). B-cells were incubated with all mitochondria-targeting reagents for 1 h prior to Tat treatment.