Hepes buffer 1 m

To prepare defined starvation medias, we reconstituted media containing the components of RPMI-1640, including the following reagents: 1XDPBS (Gibco, 14040–133), Phenol Red (Sigma-Aldrich, P3532), HEPES Buffer 1 M (Gibco, 15630–080), RPMI-1640 Vitamin Mix 100X (Sigma-Aldrich, R7256), Sodium bicarbonate (Sigma-Aldrich, S5761), reduced Glutathione (Sigma-Aldrich, G6013), 200 mM Glutamine (Gibco, 25030–081), D-Glucose (Fisher Scientific, D16), and dialyzed Fetal Bovine Serum (Sigma-Aldrich, F0392). Amino acid starvation was prepared by adding all components except amino acid mix and glucose starvation media was prepared by adding all components except glucose. See Supplemental Table 1 for full formulations. Medias were prepared, pH adjusted to 7.2–7.4, sterile-filtered, and stored at 4°C.

For the assay, the following stock solutions or reagents are to be available:

Gibco HEPES buffer 1 M (catalog#: 15630080).

Tungsten POM (phosphotungstic acid hydrate, Sigma-Aldrich); molecular weight = 2880.05 g mol⁻¹.

Molybdenum POM (ammonium heptamolybdate tetrahydrate, Sigma-Aldrich) molecular weight = 1235.86 g mol⁻¹.

NaOH 5 N solution.

Neodymium(III) acetate hydrate (99.9%), yttrium(III) acetate hydrate (99.9%), ytterbium(III) acetate hydrate (99.9%), thulium(III) acetate hydrate (99.95%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), sodium hydroxide (NaOH, ≥98%), ammonium fluoride (NH₄F, ≥99.9%), ethanol (absolute), methanol (≥99.8%), cyclohexane (≥99%), tetrahydrofuran (THF, ≥99.9%), dopamine hydrochloride (≥99.9%), hydrochloric acid (HCl, 37%), dimethyl sulfoxide (DMSO, ≥99.9%), hydroxylamine hydrochloride (HH, 99%), N,N-dimethylformamide anhydrous (DMF, ≥99.8%), nitrosyl tetrafluoroborate (NOBF₄ ≥ 95%), and PAA solution (M_w = 2000 g/mol) were purchased from Sigma-Aldrich, St. Louis, MO, USA. HEPES buffer (1 M) was purchased from Gibco. N-succinimidyl S-acetylthioacetate (SATA), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), and CD133 (Prominin-1) monoclonal antibody (12-1338-42) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. An Amicon Ultra centrifugal filter (0.5 mL, 30 K, 5 K) was purchased from Millipore (Bedford, MA, USA). Anti-EGFR antibody (ab40815), goat anti-rabbit IgG-H&L Alexa Fluor 488 (ab150077), and goat anti-rabbit IgG H&L Alexa Fluor 647 (ab150079) were purchased from Abcam, UK. A DAPI mounting kit was purchased from Vector Laboratories, Inc., Burlingame, CA, USA.

Venous blood samples were collected in EDTA-anticoagulated tubes. PBMCs were isolated with lymphocyte separation medium density gradients (Pancoll separation medium, PAN Biotech GmbH) and stored at −80 °C. Frozen PBMCs were thawed in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and 1.5% HEPES buffer 1 M (all additives from Thermo Scientific) containing 50 U ml⁻¹ benzonase (Sigma).

The human urinary bladder cell carcinoma line T24 (ATCC, HTB-4) was purchased from ATCC and was cultured in McCoy’s 5A (Modified) medium (Cat.Nr.: 16600082, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Cat.NR.:10082147, Thermo Fisher Scientific) and 1% HEPES buffer (1M) (Cat.Nr.: 15630056, Thermo Fisher Scientific) and 1% PenStrep (Cat.Nr.: 15140122, Thermo Fisher Scientific). Cells were cultured at both 1% and 21% atmosphere oxygen pressure in an InVivO2 400 hypoxic chamber (Baker Ruskinn, Sanford, MA, USA) and maintained at 37 ${}^{\circ }$ celsius, 5% CO ${}_2$ and in a humidified atmosphere. Cell irradiation was carried out using a Multi Rad 225kV irradiator. Cells were initially synchronized (see next section for protocol) prior to irradiation with 6 Gy at room temperature. The cells that received 0 Gy (control) and 6 Gy were then plated into T25 flask to allow colony formation over 10 to 12 days. A total of 220 and 7500 cells were platted per flask, respectively for the 0 Gy and 6 Gy dose values. A total of 5 flasks were made per dose point. Cell survival was evaluated using a standard colony forming assay, where colonies were counted after 10 to 12 days.

Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham, APAP, l-glutamine, l-ascorbic acid, anti-GAPDH antibody and Triton^TM X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase, insulin human recombinant and cycloheximide were purchased from Wako Pure Chemical Industries (Osaka, Japan). Soybean trypsin inhibitor powder, HEPES buffer (1 M) and Dynabeads^® Protein G were purchased from Thermo Fisher Scientific (Gibco^®; Waltham, MA, USA). 2-Mercaptoethanol, dexamethasone, Nonidet^® P-40 and polyoxyethylene sorbitan monolaurate were purchased from Nacalai tesque (Kyoto, Japan). MG132 (Z-Leu-Leu-Leu-H aldehyde) was purchased from Peptide Institute (Osaka, Japan). Proteasome substrates II, III and VI, and Fluorogenic and anti-rabbit CYP3A1 antibodies were purchased from Merck Millipore (Calbiochem^®; Darmstadt, Germany). Penicillin G potassium and streptomycin were purchased from Meiji Seika Pharma (Tokyo, Japan). Anti-mouse CYP3A1, anti-rabbit CHIP, anti-rabbit gp78-2, anti-rabbit Nrf2 and anti-mouse Ub antibodies were purchased from Santacruz Biotechnology (Dallas, Texas, USA). P450-Glo^TM CYP3A assay with luciferin-IPA was purchased from Promega (Fitchburg, WI, USA).