FTY720 down-regulates Mcl-1 expression via lysosomal degradation. RNK-16 cells were pretreated with 50nM bafilomycin A1 or 6.25nM MG132 for 30 minutes then exposed to 20μM FTY720 for 3 hours, then Western blot was performed to examine Mcl-1 expression (A) or cells were assayed for cell viability by MTT (B). (C) Dose-dependent increased expression of cathepsin B after FTY720 treatment. Western blot analysis was performed for cathepsin B in cytosolic fraction of RNK-16 cells after 24-hour treatment with 2.5, 5, and 10μM FTY720. ImageJ software was used to analyze the ratio of densitometry of cathepsin B to GAPDH as labeled in the bottom of Western blot images. (D) Confocal microscopic image of RNK-16 cells after 2 hours of treatment, showing that yellow staining indicating NBD-FTY720 (green) colocalized with cellular lysosome (top panel, red, magnification: ×63) but not mitochondria (bottom panel, red, magnification: ×40). Cellular nucleus was stained with (Hoechst 33258 blue).