Quantification of TFEB Localization

EndoC‐βH1 cells were seeded in 96‐well plates and incubated for 24 h. Cells were then treated as indicated, rinsed with PBS once, fixed for 10 min with 4% paraformaldehyde, and stained with TFEB antibodies (Cat# 4240, Cell signaling) and DAPI. For the acquisition of the images, at least 10 fields were acquired per well of the 96‐well plate by using confocal automated microscopy (Opera High Content System; Perkin‐Elmer). A dedicated script (Medina et al, 2015 (link)) was used for analysis of TFEB localization on the different images (Harmony and Acapella software; Perkin‐Elmer). The script calculates the ratio value resulting from the average intensity of nuclear TFEB fluorescence divided by the average of the cytosolic intensity of TFEB fluorescence. P‐values were calculated on the basis of mean values from independent wells.