Deta no

Mtb H37Rv was grown in 90 ml Middlebrook 7H9 medium supplemented with albumin-dextrose-catalase (ADC) and 0.05 % Tween 80 at 37 °C with shaking until OD600 was 0.4–0.5. The culture was divided into 9 equal volumes, and the cultures were treated with 10 mM paraquat (Sigma) for 1 h [66 (link)], 5 mM H₂O₂ (Sigma) for 1 h [6 (link)], 0.5 mM DETA/NO (Sigma) for 4 h [6 (link)], 3 μM MNNG (Sigma), for 1 h [24 (link)], or 20 ng/ml MMC (Sigma) for 24 h [25 (link)], respectively. Stock solutions of MNNG and DETA/NO were prepared in DMSO and 0.01 N NaOH, respectively, whereas paraquat and MMC were dissolved in sterile MQ H₂O. Untreated control cultures were prepared and analyzed in the same manner as the experimental cultures. Cells were harvested by centrifugation and frozen in RNAlater (Life Technologies). Total RNA was isolated using TRIzol (Invitrogen) followed by RNeasy spin columns (Qiagen) as previously described [64 (link)] and treated with Turbo DNase (Life Technologies) following the manufacturer’s instruction. Three independent experiments were conducted for each genotoxic stress and the controls. The workflow of methods employed to identify DE genes and non-coding RNAs are depicted in Additional file 7: Figure S2.

Petri dishes with 10 ml bottom-agar were covered with a layer of 10 ml top-agar containing 1 × 10⁸ conidia of the respective strain. In the center, 100 μl of 45 mM DETA-NO (Sigma, Germany) were filled in a hole of 10 mm diameter.

Escherichia coli DH5α was grown in LB liquid culture or agar (1.5%) supplemented with 50 μg/ml kanamycin or 250 μg/ml hygromycin B as required.
Mycobacterium smegmatis mc²155 was grown on LB agar supplemented with 25 μg/ml kanamycin or 50 μg/ml hygromycin B, and in liquid Middlebrook 7H9 medium supplemented with 10% Albumin, 0.2% glycerol, 0.02% Tween 80 and 20 μg/ml kanamycin or 50 μg/ml hygromycin B as required.
Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG were grown on Middlebrook 7H11 agar supplemented with 10% OADC, 0.4% glycerol and 20 μg/ml kanamycin or 50 μg/ml hygromycin B as required and in liquid Middlebrook 7H9 medium supplemented with 10% ADC, 0.02% glycerol and 0.02% Tween 80 in roller bottles (Cell Master, Griener Bio-One) or PETG flasks (Nalgene, Thermo Scientific), respectively. For all three mycobacterial species exponential phase cultures were harvested at 0.5 < OD₆₀₀ < 0.8. Stationary phase cultures for M. tuberculosis and M. bovis BCG were harvested at least 1 week after OD₆₀₀ = 1.0. Time course experiments cultures were harvested as indicated
Nitric oxide stress was induced by adding DETA-NO (Sigma-Aldrich) directly to cultures at a final concentration of 0.1 mM or 1.0 mM, respectively for 2 hours before RNA was isolated. To inhibit transcription initiation rifampicin was added to cultures at a final concentration of 200 μg/ml.

Mid-log phase M. smegmatis cultures (OD₆₀₀ ~ 1.2) were exposed to a single dose of H₂O₂ (Sigma Aldrich, St. Louis, USA) or the DETA/NO (Sigma Aldrich, St. Louis, USA). Doses of 0, 10, 50, 100, and 200 mM H₂O₂ and 0.05 mM DETA/NO were used. The OD₆₀₀ was monitored after exposure of the cultures to respective H₂O₂/DETA concentrations at 15-minute intervals for 2 hours, 30-minute intervals for an additional 2 h, and a final reading 5 h after oxidant exposure.

Patient-derived BT168 GSCs [50 (link),86 (link)] were cultured in serum-free medium consisting of DMEM/F12 (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 20 ng/mL EGF, 20 ng/mL bFGF, 2% B27 w/o vitamin A (Thermo Fisher Scientific, Waltham, MA, USA), 1% glutamine and 1% antibiotics (Sigma-Aldrich) in an incubator under standard culture conditions. GSC proliferation generated floating rounded spheres with well-defined borders, which were kept until they were suitably sized to ensure cell health, and well spaced from each other to avoid sticking. Twice a week, subconfluent spheres were centrifuged at 400 rpm for 20 min and then incubated with accutase (Thermo Fisher Scientific) for 15 min at 37 °C. Single GSCs were centrifuged at 800 rpm for 5 min and seeded in fresh medium. U87MG cells (from the American Type Culture Collection, Manassas, VA, USA) were routinely grown in DMEM supplemented with 10% FBS, 1% glutamine, and 1% antibiotics (Sigma-Aldrich). DETA/NO, TMZ and DMSO were from Sigma-Aldrich. DETA/NO spontaneously releases NO in aqueous media with a half-life of 20 h. Therefore, it was dissolved in sterile water and supplied to the cells every 48 h. TMZ was dissolved in DMSO and supplied every 72–96 h. Parallel to TMZ treatment, control cells were always supplied with DMSO, diluted as TMZ.

During this study the following drugs were used: diethylenetriamine/nitric oxide adduct (DETA-NO, 250–500 µM, Sigma), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 500 µM-1 mM, Sigma), 8-(4-Chlorophenylthio)-guanosine 3′, 5′-cyclic monophosphate sodium salt (8-pCPT-cGMP, 500–750 µM, Sigma), 1 H-[1] (link), [2] (link), [4] (link)Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 500 µM, Ascent Scientific), tetrodotoxin (TTX, 1 µM, Ascent Scientific), (+)-tubocurarine hydrochloride (tubocurarine, 3 µM, Sigma), formamide (2 M, Sigma) and 18-β-glycyrrhetinic acid (18βGA, 100 µM, Sigma).

MafB/c-Maf double deficient (Maf-DKO) macrophages were a kind gift from Dr. Michael H. Sieweke (Center d'Immunologie de Marseille-Luminy). Maf-DKO cells were grown in DMEM containing 20% L929-conditioned medium and 10% FCS. BMDM were differentiated from bone marrow cells obtained from C57BL6 mice in the presence of DMEM containing 20% L929-conditioned medium and 10% FCS. Maf-DKO cells were predominantly octaploid (8c) whereas BMDM were diploid (2c) and octaploid (8c) as determined by DNA quantification with DAPI staining (data not shown). For experiments, cells were incubated for 24 h at 37°C and 5% CO₂ in low glucose, low glutamine medium (henceforth LGLG medium) which contained DMEM, 44 mM sodium bicarbonate, 10% FCS, glucose (4.8 mM), and glutamine (0.8 mM) with or without IFNγ 10 ng/mL or IL-4 10 ng/mL (both from Preprotec), and C75, etomoxir, triacsin, oligomycin, DETA/NO, or SEITU (all from Sigma) at the indicated concentrations. In a set of experiments, FCS was replaced with delipidated serum (Lipoprotein deficient serum from fetal calf, Sigma) plus a 1:100 dilution of lipid mixture (Lipid mixture 1, Sigma) containing cholesterol, and arachidonic, linoleic, linolenic, myristic, oleic, palmitic, and stearic acids.