Expression of secretory pathway Spn4A.RRLL(s) in Huh-7.5.1 cells dramatically reduces the abundance of cellular lipid storage droplets LDs are dynamic intracellular lipid storage compartments made up of triglyceride and cholesterol esters, surrounded by a phospholipid membrane and associated with specific marker proteins including adipose differentiation-related protein (ADRP), also known as perlipin 2 [74] . Because SREBP activation controls the expression of genes directly involved in intracellular fatty acid and cholesterol biosynthesis (reviewed in [36] ) and because cholesterol-ester levels were reduced by Spn4A.RRLL(s), we investigated the effect of serpin-mediated SKI-1/S1P inhibition on cellular LD abundance. Fluorescence microscopy was used to determine the relative abundance of LDs stained with BODIPY 493/503 in Huh-7.5.1 cells infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), and Ad-Spn4A.RRLL(s). After 72 hours, the level of BODIPY-stained LDs in Spn4A.RRLL(s)-expressing cells was dramatically reduced compared to empty vector-treated cells ( Figure 4A ). By contrast, Spn4A.RRLL(r)-expressing cells had no apparent reduction in LD size or abundance compared to controltreated cells ( Figure 4A ). Quantification of confocal microscopy images demonstrated that, on average, LD abundance was reduced by 80% in Spn4A.RRLL(s)-expressing cells compared to controls ( Figure 4B ). The effect of Spn4A.RRLL(s) expression on cytosolic LD abundance was confirmed by visualizing the LD marker ADRP/perilipin 2 using confocal microscopy ( Figure 4C ) and by using quantitative Western blot ( Figure 4D ). Spn4A.RRLL(s) was observed to reduce ADRP/perilipin 2 protein expression by 50% compared with control cells ( Figure 4D ), whereas there was no reduction in ADRP/perlipin 2 expression following Spn4A.RRLL(r) treatment. We subsequently confirmed that reduced cellular LD levels were due to inhibition of SKI-1/S1P using 10 mM PF-429242 whereupon ADRP/perilipin 2 levels were reduced by 62% compared to control DMSO-treated cells ( Figure 4E ). These results confirm that Spn4A.RRLL(s)-and PF-429242-mediated inhibition of SKI-1/S1P enzymatic activity dramatically reduces intracellular LD abundance in Huh-7.5.1 cells.