Mestrenova v 10

All NMR spectra were recorded at the at the University of Denver on a Bruker UltraShield 500/54 Plus spectrometer. All Spectra were processed and analyzed using TopSpin v. 2.1 program (Bruker). All peptide NMR samples were prepared in 99.96% deuterated water. Water Suppression signal was applied at a frequency of 2353.37 Hz for all ¹H NMR spectra. Signals were integrated, and the coupling constants were calculated in MestReNova v. 10.0.1 program (Mestrelab Research).

Cells were harvested, washed twice in 1× PBS (Life Technologies) and quantified using Trypan blue (#T8154, Sigma-Aldrich) exclusion assay (three independent counts). A minimum of 7 × 10⁶ cells per sample were used for each extraction and total cell numbers were used to normalize between MRK003 and DMSO counterparts. The methanol–chloroform–water (1/1/1, v/v/v, all Sigma-Aldrich) dual-phase cell extraction protocol was applied to obtain water- and lipid-soluble metabolites as previously described.^{19 (link)} The lyophilized water-soluble extracts were resolved in 495 μl deuterium oxide (D₂O, #151882, Sigma-Aldrich) supplemented with 5 μl D₂O containing 0.05% 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid (TSP, #450510, Sigma-Aldrich) as internal concentration standard. The extracts were analyzed in the Department of Radiology, Johns Hopkins Hospital on a Bruker Avance 500 spectrometer operating at 11.7 T using a 5-mm HX inverse probe run at 25°C as previously described.^{19 (link)} The fully relaxed ¹H-NMR data were postprocessed and metabolites were quantified through peak integration using Mestrenova v10.0 (Mestrelab Research, Santiago de Compostela, Spain, CA). For each sample, Notch activation status was assessed by quantification of canonical target gene expression.