4 nitroquinoline n oxide 4 nqo

E. coli DH10B strain (Invitrogen, Waltham, MA, United States) was routinely grown in Luria-Bertani (LB) medium (Condalab, Madrid, Spain) at 37°C. The growth medium for transformed E. coli DH10B strains was supplemented with 100 μg mL^–1 ampicillin (LB-Ap) to maintain the pBlueScript II SK (+) plasmid (pSKII+). When required, working concentrations of 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal) and isopropyl-β-D-1-thiogalactopyranoside (IPTG) were 40 μg mL^–1 and 100 μM, respectively. For solid cultures, the growth medium was supplemented with agar (15 g L^–1). Liquid cultures were shaken on an orbital platform operating at 200 r.p.m. For resistance assays, growth medium was supplemented with the minimal inhibitory concentration (MIC) of the different agents: 125 mM sodium perchlorate (NaClO₄) (Thermo Fisher Scientific, Waltham, MA, United States), 0.2 μM 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich) or 1 mM hydrogen peroxide (H₂O₂) (Sigma-Aldrich, St. Louis, MO, United States). MICs were established as the lowest sublethal concentration of the different toxic compounds for E. coli DH10B carrying empty pSKII+ vector (DH10B/pSKII+).

All stock solutions were prepared in DMSO, except FB1 which was dissolved in water. The selected maximum concentration to be tested in the SOS/umu assay was 40 mg/mL (corresponding to 1000 µg/mL in the 96-well plate). For insoluble substances, the highest soluble concentration was used. Insolubility was assessed as precipitation (or turbidity) in the solvent and evident to the unaided eye over a dark background. For very soluble mycotoxins (NIV, 3ADON, 15ADON, T-2, HT-2) that were not toxic for bacteria and induced an equivocal response, two extra concentrations were also tested: 160 and 80 mg/mL (corresponding to 4000 and 2000 µg/mL in the 96-well plate, respectively). The positive control stock solutions were prepared in DMSO: at 500 µg/mL (12.5 µg/mL in 96- well plate) for 2-aminoanthracene (2-AA) (Sigma-Aldrich, Germany) and at 100 µg/mL (2.5 µg/mL) for 4-nitroquinoline-n-oxide (4-NQO) (Sigma-Aldrich, China). 4NQO was the positive control without metabolic activation (PBS) and 2AA with metabolic activation (S9).

DMSO and Na₂CO₃ were purchased from PanReac AppliChem (Barcelona, Spain). Bactotryptone for TGA medium was obtained from Bectone Dickinson (Madrid, Spain) and dextrose and NaCl were from PanReac AppliChem (Barcelona, Spain). Ampicillin, ONPG (2-nitrophenyl- β-D-galactopyranoside), the B-buffer ingredients (Na₂HPO₄ 2H₂O, NaH₂PO₄ H₂O, MgSO₄ 7H₂O, sodium dodecyl sulfate, β-mercaptoethanol) in which the ONPG was dissolved, PBS ingredients (Na₂HPO₄ 2H₂O, NaH₂PO₄ H₂O) and the positive controls 2-aminoanthracene (2AA) and 4-nitroquinoline-N-oxide (4NQO) were purchased from Sigma-Aldrich (Darmstadt, Germany). The KCl for B-buffer was from PanReac AppliChem (Barcelona, Spain). Ingredients for the S9 mix preparation were obtained from Sigma-Aldrich (Darmstadt, Germany)—phosphate buffer (NaH₂PO₄ H₂O, Na₂HPO₄ 2H₂O), glucose-6-phosphate and NADP solutions—or from PanReac AppliChem (Barcelona, Spain)—saline solution (MgCl₂ 6H₂O, KCl).

4-Nitroquinoline N-oxide (4-NQO; Sigma) was diluted in DMSO to a final concentration of 50 mM, aliquoted, sealed, and stored at −80°C. UV irradiation was performed in Crosslinker CL-1000 using 254 nm wavelength lamp with dose 40-60 J/m². Flavopiridol (Sigma) and 4-Thiouridine (4sU; Carbosynth) were diluted in DMSO to a final concentration of 1 mM and stored at −20°C. Tetracycline hydrochloride (Sigma) was diluted in water to a final concentration of 1 mg/ml and stored at −20°C. SB203580 (Selleckchem) was diluted in DMSO to a final concentration of 50 mM and stored in −20°C.

Strain construction, cell growth, yeast transformation, spotting assay followed standard protocols (Guthrie and Fink, 2002 ). Yeast knockout and TAP-tagged strains were obtained from Open Biosystems (GE Healthcare Dharmacon Inc., France). Other strains are listed in Supplementary file 3. Cell irradiation with UV light was performed with calibrated 254 nm UVC germicidal lamp. Cell treatment with 0–0.2 μg/ml 4-Nitroquinoline N-oxide (4-NQO, Sigma–Aldrich, France) was performed in log-phase cultures for 3 hr before analysis. For experiments with proteasome inhibitor MG132 cells were grown on synthetic medium containing L-proline instead of ammonium sulfate as the sole nitrogen source and were permeabilized with sodium dodecyl sulfate (SDS, 0.003%) before addition of the inhibitor (75 μM final concentration for 3 hr) (Liu et al., 2007 ). Genotoxic treatment and colony-forming assays were performed as described previously (Liu et al., 2002 (link)).

For the SOS/umu test, GE was dissolved in water at 40 mg/mL. This concentration was selected based on the extract’s solubility in water (the extract showed some precipitation when prepared at 80 mg/mL). The final concentration in the 96-well plates (plate B, see Section 2.4.2) was 1 mg/mL (1/40 dilution).
The positive control stock solutions were prepared in DMSO at 0.5 mg/mL (corresponding to 0.0125 mg/mL in 96-well plate B) for 2-aminoanthracene (2-AA) (Sigma-Aldrich, Taufkirchen, Germany) and at 100 µg/mL (2.5 µg/mL in 96 well-plate B) for 4-nitroquinoline-n-oxide (4-NQO) (Sigma-Aldrich, Beijing, China). 4NQO was the positive control without metabolic activation (PBS) and 2AA with metabolic activation (S9). Water was used as the negative control.