Apilimod (HY-14644) was purchased from MedChem Express (Monmouth Junction, NJ). Aliquots were prepared in ultra-pure grade dimethyl sulfoxide (DMSO) (VWR; 97063-136) and stored at −20°C until use. On each experimental day, a fresh aliquot of Apilimod (200 μM) was thawed. At 24 hours after transfection, cells were treated with either Apilimod (final concentration, 200 nM) or DMSO (1:2000, v/v) for 1 to 1.5 hours at 37°C. Cells were prepared for live cell imaging. As a positive control for Apilimod treatment, the diameter of lysosomal-associated membrane protein 1 (LAMP-1)–positive intracellular vesicles was measured and found to be substantially increased in treated compared to untreated cells (2.2. μm versus 1.7 μm), which is consistent with previous reports (fig. S2L) (31 (link)).