Filipin staining of haploid models of NPC1 variants. Wild-type (WT) HAP1 cells (top left) displayed no defect in cholesterol trafficking. Pathogenic variants, NPC1 p.I1061T and NPC1 p.Y634C (top right and bottom left, respectively), show distinct foci of filipin staining indicative of a defect in cholesterol trafficking. NPC1 p.L785S (bottom right) was found to show a biochemical variant phenotype, with less definitive filipin staining. White dashed-bordered box has been enlarged twofold and inset at bottom right. Scale bars, 13 μm.