Vnmrj 3

Volumes of autocorrelation and cross-peaks in ¹H-¹⁵N NzExHSQC spectra were integrated using VNMRJ 3.2 (Varian Inc.) software. The volume of autocorrelation peak O₅ at a mixing time of 13 ms was set to 100, and all other integrals were normalized to this value. Exchange rates were determined using iterative least-squares fitting, performed in Origin 8.1 (OriginLab, Northampton, MA, USA). The decrease of intensity of the autocorrelation peaks B, O₅, I, and O₃, as a function of the mixing time (τ_m), is a result of ion movement from the original binding site as well as the spin-lattice relaxation (T₁), and is best described by the bi-exponential function [44 (link),46 (link)]: $$V_{auto}\left({\tau }_m\right)=A_1\left[e^{-r_1{\tau }_m}\right]+A_2\left[e^{-\frac{{\tau }_m}{T_1}}\right]$$
where A₁ and A₂ are scaling factors and r₁ is a rate constant. Estimated T₁ relaxation times of autocorrelation peaks B and I were used in the analysis of cross-peak volumes as a function of the mixing time, corresponding to ¹⁵NH₄⁺ ions exchanging with the bulk solution and ions moving within the [d(G₄C₂)]₄ G-quadruplex, respectively. Cross-peak volumes as a function of the mixing time were fitted to the following equation [44 (link),46 (link)]: $$V_{cross-peak}\left({\tau }_m\right)=A\left[e^{-\frac{{\tau }_m}{T_1}}\right(1-e^{-k{\tau }_m}\left)\right]$$
where A is a scaling factor and k is the exchange rate.

2D 1H J-resolved (JRES) NMR spectra were acquired on a 500 MHz Varian/Agilent spectrometer (Agilent, Santa Clara, CA) using a double spin echo sequence with 4 transients per increment for a total of 32 increments. These were collected into 16 k data points using spectral widths of 6 kHz in F2 and 40 Hz in F1. There was a 2.0 s relaxation delay. Each FID was Fourier transformed after a multiplication with sine-bell/exponential function in the F2 dimension and a sine-bell function in the F1 dimension. JRES spectra were tilted by 45°, symmetrised about F1, referenced to TSP at dH = 0.0 ppm and the proton-decoupled skyline projections (p-JRES) exported using Agilent VNMRJ 3.2 software. Metabolites responsible for the separation between samples from cells cultured in SMG or NG condition were identified using an in-house NMR database and Chenomx NMR suite v. 7.7 (Chenomx Inc., Alberta, Canada).

Each medium sample (2 ml) was lyophilized and then dissolved in 700 μl of 1 mM TSP [sodium salt of 3 (trimethylsilyl) propionic-2,2,3,3-d4 acid], 10 mM sodium azide D₂O phosphate buffer solution (pH = 7.4) and finally homogenized by vortex mixing for 1 min. After centrifugation (10 min, 10.000 RCF at 22 °C), 600 μl of each resulting supernatant was transferred to a 5-mm NMR tube and used for the NMR analysis2D. 1H J-resolved (JRES) NMR spectra were acquired on a 500 MHz Varian/Agilent spectrometer (Agilent, Santa Clara, CA) using a double spin echo sequence with 4 transients per increment for a total of 32 increments. These were collected into 16 k data points using spectral widths of 6 kHz in F2 and 40 Hz in F1. There was a 2.0 s relaxation delay. Each FID was Fourier transformed after a multiplication with sine-bell/exponential function in the F2 dimension and a sine-bell function in the F1 dimension. JRES spectra were tilted by 45°, symmetrised about F1, referenced to TSP at δH = 0.0 ppm and the proton-decoupled skyline projections (p-JRES) exported using Agilent VNMRJ 3.2 software. Metabolites responsible for the separation between samples from cells treated with hypoxia in the presence or absence of siChe-1 were identified using an in-house NMR database and Chenomx NMR suite v. 7.7 (Chenomx Inc., Alberta, Canada).

2D ¹H J-resolved (JRES) NMR spectra were acquired on a 500 MHz Varian/Agilent spectrometer (Agilent, Santa Clara, CA) using a double spin echo sequence with 4 transients per increment for a total of 32 increments. These were collected into 16 k data points using spectral widths of 6 kHz in F2 and 40 Hz in F1. There was a 2.0 s relaxation delay. Each FID was Fourier transformed after a multiplication with sine-bell/exponential function in the F2 dimension and a sine-bell function in the F1 dimension. JRES spectra were tilted by 45°, symmetrised about F1, referenced to TSP at d_H = 0.0 ppm and the proton-decoupled skyline projections (p-JRES) exported using Agilent VNMRJ 3.2 software. Metabolites responsible for the separation between treated and untreated samples were identified using an in-house NMR database and Chenomx NMR suite v. 7.7 (Chenomx Inc., Alberta, Canada).

Data were acquired in a 9.4 T horizontal-bore magnet (Magnex Scientific, Abingdon, United Kingdom) with a VNMRS console (Varian, Palo Alto, CA, United States) and VnmrJ 3.2 (Agilent Technologies, Santa Clara, CA, United States). A surface coil equipped with a single loop tuned to ¹H (400.2 MHz) and two 16 mm loops in quadrature tuned to ¹³C (100.67 MHz) was placed over the left kidney (to avoid signals from the liver), its position verified by ¹H GEMS MRI, and shimmed using FASTESTMAP (Gruetter and Tkác, 2000 (link)). A series of ¹³C magnetic resonance spectra were acquired starting with the pyruvate infusion (∼2.9 s repetition time, with respiratory gating and cardiac triggering, 30° BIR4 adiabatic excitation, 20161.3 Hz spectral width, 8258 complex points, WALTZ-16 ¹H decoupling).

2,2’-azobisisobutyronitrile (AIBN) and pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Dipentaerythritol hexakis(3-mercaptopropionate) (DPEHMP) was purchased from TCI America (Montgomeryville, PA, USA). Methacryloxyethyl thiocarbamoyl rhodamine B was purchased from Polysciences (Warrington, PA, USA). Trifluoroacetic acid (TFA) and solvents were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). tert-Butyl acrylate, methyl acrylate, and methyl mercaptopropionate (MMP) were purchased from Acros Organics (Morris County, NJ, USA). The inhibitors of these monomers were removed by passing through alumina before use. Other chemicals and solvents were used without further purification. 1H NMR was performed using a Varian MR400 (400 MHz, Agilent Scientific Instruments, Santa Clara, CA, USA) and analyzed using VNMRJ 3.2 (Agilent Scientific Instruments, Santa Clara, CA, USA) and MestReNova. Gel permeation chromatography (GPC) analysis was performed using a Waters 1515 HPLC instrument (Milford, MA, USA) using THF as an eluent, equipped with Waters Styragel (7.8 × 300 mm) HR 0.5, HR 1, and HR 4 columns in sequence and detected by a differential refractometer (RI). Sintered HAP discs (0.5 cm in diameter) were purchased from Himed, Inc. (Old Bethpage, NY, USA).