Filipin III staining

HT1080 cells were seeded onto coverslips, grown to 50% confluency, and incubated at 37°C for 4h with inhibitor or vehicle in serum free MEM. Cells were fixed with formalin, incubated with Filipin III (50 μg/mL in PBS) for 2h at RT, and coverslips mounted with PermaFluor Aqueous mounting medium. Cells were imaged by confocal microscopy (LSM800 AxioObserver Z1, Zeiss) using a 63x / 1.4NA oil Plan Apochromat objective. An average of twenty z-stacks were acquired per image, with a pixel size of 0.1 μm.