Additional file 1 of Detection of glucose-derived d- and l-lactate in cancer cells by the use of a chiral NMR shift reagent
Creators: name: Suh, Eul Hyun
name: Geraldes, Carlos F. G. C.
name: Chirayil, Sara
name: Faubert, Brandon
name: Ayala, Raul
name: DeBerardinis, Ralph J.
name: Sherry, A. Dean
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Computational Biology

description: Additional file 1: Figure S1. Titration curves for d- and l-lactate. Figure S2. Full 1H NMR spectrum of d-lactate (15 mM) and l-lactate (15 mM) addition to Yb3 (6.3 mM) showing the presence of excess free lactate CH3 and CH resonances in their normal diamagnetic positions (inserted). Figure S3. Detection of d- and l-lactate formation in erythrocytes using Yb3 (2.2 mM) by 1H NMR spectra of the supernatant of erythrocytes (40 % hematocrit) in phosphate buffered saline, pH 7.4 after incubation with (a) 5 mM glucose for 2 h (b) 5 mM methyl glyoxal for 2 h (c) 5 mM glucose for 30 min followed by addition of 5 mM MG for additional 1.5 h and (d) 5 mM methyl glyoxal for 2 h (d) wo/substrate for 2 h at 37°C. Figure S4. Detection of d- and l-lactate formation in erythrocytes at each time point during 2 h incubation using Yb3 (2.2 mM) by 1H NMR spectra of the supernatant of erythrocytes (40 % hematocrit) in phosphate buffered saline, pH 7.4 (a) 5 mM glucose for 2 h (b) 5 mM glucose for 30 min followed by addition of 5 mM MG for additional 1.5 h and (C) 5 mM methyl glyoxal for 2 h (d) wo/substrate for 2 h at 37°C. The data was normalized with no substrate incubation data as background correction. Figure S5. CEST spectra of the Yb3 complex (5mM) in RBC cultured media. Presaturation pulse of 5s with B1 of 15uT was applied at 298K using 9.4 NMR spectrometer: RBCs (40% hematocrit) was incubated in Phosphate-buffered saline (pH ~7.0, 2 h at 37 °C) with 5mM glucose; Glc 5mM, 5mM glucose 30min incubated then add 5mM methyl glyoxal; Glc + MG, and 5mM methyl glyoxal; MG 5mM. The CEST peaks are assignable at 168ppm to d-lactate and at 157ppm are l- lactate. Figure S6. 1H NMR (400MHz) spectra of media collected from (a) sgGlo1 pLHCX cells (re-expressed GLO1) (b) sgGlo1 (GLO1 deleted cells) and (c) parental 3353T3 cells. Each cell line was incubated with 5mM glucose in DMEM basal media for 4 h, 37 °C, pH 7. Subsequently, a 0.5 mL volume of media was collected and mixed with 10 mM Yb3 prior to collection of the NMR spectra. The highlighted methyl resonances are assigned to l-(blue) and d-(red) lactate. Figure S7. 1H NMR spectra of an aqueous solution of Yb3 (6 mM) in the presence of d-lactate (15 mM) and l-lactate (15 mM) at pH 6.0 and 7.4. The chemical shifts of CH3-L-lac and CH3-D-lac were insensitive to pH over this range. The signals of excess unbound d- and l-lactate appeared in their normal diamagnetic positions (not shown here). Table S1. 1H NMR shift (ppm) of l-lactate·Yb3 and d-lactate·Yb3 of RBCs supernatant sample.