Miglustat

Thapsigargin, filipin, CaCl₂, EDTA, and cholesterol were purchased from Sigma. EGTA-AM was purchased from Thermo Scientific. Dynasore was purchased from Abcam. Miglustat was purchased from Tocris. Antibodies used in the study were: mouse anti-α-tubulin (Abcam); rat anti-LAMP1 (Clone 1D4B, Development Studies Hybridoma Bank), mouse anti clathrin heavy chain (BD Biosciences), mouse anti-STIM1 (Cell Signalling), rabbit anti-TFEB (Proteintech), rabbit anti-calcineurin (Abcam), rabbit anti-histone H3 (Cell Signalling), and rabbit anti SREBP (Abcam). For immunoblotting, the secondary antibodies were conjugated to horseradish peroxidase (Jackson Laboratories) or fluorochromes (IR-dye 800 or IR-dye 680; LI-COR). Secondary antibodies used for immunofluorescence were purchased from Thermo Scientific.

Miglustat (Tocris, Ellisville, MO, USA) is less effective in GCS inhibition than Genz. Therefore, a higher concentration in the medium (100µM in H₂O) has been used to decrease GSLs in a significant manner [69 (link)]. Hepa 1-6 cells were cultivated in analogy to the Genz treatment.

Small-molecule inhibitors were added to cell culture medium 1 h prior to stimulation. ML-130 (5 µM) and ponatinib (50 nM) were purchased from Selleck Chemical. Compound 21a (XIAP-Cp-21a, 1–2.5 µM) was kindly provided by TetraLogics Pharmaceuticals.26–28 (link) To induce the lysosomal lipid storage phenotype in MDM/PBMC, cells were pre-incubated with 2 µg/mL U18666A (Sigma-Aldrich) for 48/24 h. Other compounds were bafilomycin A1 (50 nM, Enzo Life Sciences), 2-hydroxypropyl-β-cyclodextrin (0.5–2%, Sigma-Aldrich), chlorpromazine (1–10 μg/mL, Merck Millipore), rapamycin (1–10 µM, Cayman Chemical), Torin 1 (10 µM, Tocris), d-(+)-trehalose dihydrate (100 mM, Sigma-Aldrich) and miglustat (4–400 µM, Tocris).