Filipin staining

Folch extraction was performed on 70 μg of protein from PHT lysate as described^{38 (link)}. Lysates were incubated with methanol/chloroform (1:2 v/v, 30 min, 50 °C). Then, 1 volume of water was added (18 h, 4 °C), and the reaction was centrifuged (750 RCF, 20 min, 4 °C). The methanol/water phase was removed, and the chloroform phase containing cellular cholesterol was recovered and completely evaporated under nitrogen. Cholesterol extracted from PHT was determined with the Amplex Red Cholesterol assay (Invitrogen, USA) in the presence or absence of the enzyme cholesterol esterase for determination of total (TC) and free cholesterol (FC), respectively. Cholesterol esters were determined as the difference between TC and FC. FC was also determined by Filipin staining (Sigma-Aldrich, USA) as described^{39 (link)}. PHT cells were fixed with 4% paraformaldehyde, their autofluorescence was quenched with glycine in PBS (1.5 mg/mL, 20 min, 20 °C), and then they were incubated with Filipin (25 μg/mL, 30 min, 20 °C). Images were obtained in an EVOS FL Imaging System (Life Technologies, USA), and the fluorescence was quantified with ImageJ version 1.48 (NIH, USA).