2.5.2. TFEB Nuclear Translocation Assay

MEC-1 cells (1 × 10⁶) were incubated with indicated compounds or cultured in Hank’s Balanced Salt Solution (HBSS; Gibco, Grand Island, NY, USA) for 1 h. The cells were then centrifuged (200× g, 5 min) and fixed with 0.5 mL 4% paraformaldehyde for 10 min. There followed a wash with 0.5 mL PBS with centrifugation as before, and permeabilization with 0.5% Triton X-100 in 5% bovine serum albumin (BSA)/PBS for 20 min. Subsequently, cells were washed and incubated with 100 µL 5% BSA/PBS for 30 min. To this, 1 µL Anti-TFEB Antibody (C-6) (1:100 dilution; sc-166736; Santa Cruz Biotechnology, Dallas, TX, USA) was added and incubated at room temperature for 1 h. The cells were washed again and resuspended in 100 µL 5% BSA/PBS, to which 0.2 µL goat anti-mouse IgG-PE antibody (1:1000 dilution; sc-3738; Santa Cruz Biotechnology, Dallas, TX, USA) was added and incubated in the dark for 1 h. Then, 100 µL 6 µM DAPI was added (final concentration, 3 µM), incubated for 5 min, then the cells were washed with 0.5 mL PBS. The pellet was resuspended in 20 µL PBS and a minimum of 5,000 events in focus was collected using an imaging flow cytometer (Amnis ImageStream X Mk II; Luminex Corporation, Austin, TX, USA). The basal level of nuclear translocation of TFEB in control cells was determined by comparison of the DAPI and TFEB images using the IDEAS wizard Nuclear localization. Gating on control cells with translocated TFEB was used as the template for batch analysis of the other samples. The data are presented as mean proportions of cells with TFEB translocated to the nucleus ± SEM, from ≥3 independent experiments.