Oxaloacetate

Cell culture grade DMSO (Cat No D2650), Congo Red (Cat No C6277), Dulbecco’s phosphate buffered saline (DPBS, D8537), Evans Blue (Cat No E2129), β-nicotinamide adenine dinucleotide (NADH, Cat No N4505), oxaloacetate (Cat No O4126) and RPMI 1640 cell culture media (Cat No R8758) were purchased from Sigma-Aldrich. Fulvestrant (Cat No S1191) was purchased from Selleckchem. Lapatinib (HY-50898), nilotinib (Cat No HY-10159), sorafenib (Cat No HY-10201) and vemurafenib (Cat No HY-12057) were purchased from MedChem Express. 3′,3′′,5′,5′′-Tetraiodophenolphthalein (TIPT, Cat No sc-216639) was purchased from Santa Cruz Biotechnology. AmpC β-lactamase was purified from Escherichia coli as previously described.^{30 (link)} Malate dehydrogenase (MDH, Cat No 442610) and CENTA (Cat No 219475) were purchased from EMD Millipore. Trypsin (Cat No T0303) and Suc-Ala-Ala-Pro-Arg-pNA (Cat No L1720) were purchased from Sigma-Aldrich and BACHEM respectively. Cell line MDA-MB-231 (Cat No HTB-26) was purchased from ATCC. Triton X-100 (Cat No 21568-2500) was purchased from Acros. Presto Blue cell viability reagent (Cat No A13262) was purchased from Invitrogen. 96-well plates (Cat No 655096) for DLS were purchased from Greiner Bio-One.

Activity assays were performed using a citrate synthase activity assay kit (Sigma CS0720, St. Louis, MO, USA) per the manufacturer’s instructions. Briefly, skeletal muscles or primary myotube lysates were generated by glass pestle homogenization by hand in RIPA buffer. Protein concentrations were determined using a BCA Protein Assay (Pierce Thermo Fisher #23225, Waltham, MA, USA). Activity assays were performed in assay buffer containing (in mmol/L): 100 Tris, 1 EDTA, 1 EGTA, 10 DTNB (Sigma: D8130), and 30 Acetyl-CoA at pH 8.35. All samples were measured in duplicate and the average absorbance was used in final calculations of activity. Background absorbance was measured prior to addition of 10 mM oxaloacetate (Sigma: O4126) and final activity rates were corrected for those values.