HepG2 cell line was treated with GNS561 (1, 5 and 10 µM), HCQ (50, 100 and 200 µM) and HDSF (25 and 100 µM) for 3 h. The cells were lysed in 0.5% Triton X-100 with cOmplete™ Protease Inhibitor Cocktail. The cell lysates were used a source of PPT1 and PPT1 activity was assayed using 4-methylumbelliferyl-β-D-6-thiopalmitoyl-glucoside as reported [37 (
link)]. Reaction mixtures contained 5 µL of cell lysate + 5 µL 0.5% Triton X-100 with cOmplete™ Protease Inhibitor Cocktail + 20 μL of substrate preparation (0.5 mM substrate, 1.5 mM dithiothreitol, 0.1 U
almond β-glucosidase (Sigma-Aldrich, G4511), 0.2% Triton X-100 and McIlvain’s phosphate citrate buffer (0.2 M Na
2HPO
4, 0.1 M citric acid, pH 4). After 1 h incubation at 37°C, the reaction was stopped by adding 200 µl of 0.5 M glycine-NaOH, pH 10.5 buffer. The amount of the released fluorescent product 4-methylumbelliferone was determined by fluorometry at 358 and 448 nm for the excitation and emission wavelengths, respectively. Infantile subtype of ceroid lipofuscinosis fibroblasts which contain
PPT1 mutations and normal fibroblasts were used as control. 4-methylumbelliferone diluted in 0.5 M glycine-NaOH, pH 10.5 buffer was used to do a standard curve and to calculate the enzymatic activity of PPT1.
Brun S., Bestion E., Raymond E., Bassissi F., Jilkova Z.M., Mezouar S., Rachid M., Novello M., Tracz J., Hamaï A., Lalmanach G., Vanderlynden L., Legouffe R., Stauber J., Schubert T., Plach M.G., Courcambeck J., Drouot C., Jacquemot G., Serdjebi C., Roth G., Baudoin J.P., Ansaldi C., Decaens T, & Halfon P. (2021). GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions. Autophagy, 18(3), 678-694.
