Fig. (2) Experimental design. Huh7 cells were plated and transfected with the PCSK9-HaloTag fusion gene to establish a stable cell line expressing PCSK9-HT. To label secreted PCSK9-HT, 0.2 µM of Alexa Fluor 488 ligand was incubated with cells in DMEM media supplemented with 10% FBS for various times, from 30 min, 60 min, 120 min, 180 min, 240 min and 300 min to overnight, followed by fixation and permeabilization. Intracellular PCSK9-HT was labeled with 5 µM of TMR ligand for 15 min. The cells were fixed and permeabilized at various time points, from 30 min, 60 min, 120 min, 180 min, and 240 min to 300 min. The cells were counter-stained with LDLR and subcellular organelle markers. Co-localization of internalized or intracellular PCSK9-HT with LDLR and subcellular organelles was visualized by confocal fluorescence imaging.