Figure 5

Loss of HSulf-1 induced enhanced β-oxidation and lipolytic enzymes. (A) The samples were extracted using Metabolon's standard solvent extraction method from cells in logarithmic phase having five biological replicates for each sample and distributed into equal parts for analysis on the GC/MS and LC/MS/MS platforms. Fold increase of carnitine and its derivatives were calculated by the average metabolite level of Sh1/NTC and Sh2/NTC. *p = 0.01 to 0.09; **p = 0.001 to 0.009; ***p < 0.001 compared to NTC. (B). Levels of CPT1A in NTC, Sh1, and Sh2 cells determined by real-time PCR and Western blot analysis. *p < 0.05; **p < 0.01; ***p < 0.001 compared to NTC. (C) FAO in terms of OCR in pmol/min/mg of protein was monitored using a Seahorse Bioscience Extracellular Flux Analyzer in real time (mean ± S.D., n = 3). Cells treated with etomoxir (50 μM), an inhibitor of carnitine palmitoyltransferase 1, served as a positive control. Changes in the FAO induction in Sh1 and Sh2 cells are compared with that of NTC cells. Etomoxir-induced inhibition of FAO in Sh1 and Sh2 cells are compared with the FAO inhibition in NTC (p ≤ 0.01 and p ≤ 0.001). (D) Immunoblot analysis of MAGL, DAGLA, HSL, ASCL1 in NTC, Sh1, and Sh2 where tubulin used as internal loading control.