Fig 2: Assessment of PCSK9 and LDLR expression in human LEC. (A) PCSK9 expression was measured by immunoblotting in either human LEC, HepG2 or HEK 293T cell lysates. (B) ELISA was used to measure PCSK9 levels in the cell culture supernatant of Huh7 (grey), HEK 293T (white) and human LEC (black). (C) Expression of LDLR was detected in protein lysates by immunoblotting of human LEC. Huh7 cells were used as a positive control and HEK 293T cells treated with siLDLR were used as a negative control. LDLR protein expression on human LEC was measured by (D) flow cytometry after extracellular staining of LEC (black line) and Huh7 (grey line) and by (E) immunofluorescence (Blue, DAPI; red, cholera toxin; green, anti-LDLR; yellow, colocalized voxels; scale bar, 20 µM). (F) Scatterplot of red and green pixel intensities of cholera toxin (red) and anti-LDLR (green) in human LEC. n = 4-9. Statistics: ****p < 0.0001.

Fig 3: LDLR regulation in human LEC. (A) Cell surface expression of LDLR was measured by flow cytometry after incubating human LEC with (ctl) or without FBS-containing media for 24 h. (B) Human LEC were incubated with Dil-LDL for 4 h and LDL uptake was measured by flow cytometry (dotted line, no LDL; grey line, Dil-LDL). (C) The percentage of LDL internalization compared to control as well as (D) cell surface expression of LDLR was then quantified by flow cytometry. Human LEC were incubated with exogenous human recombinant PCSK9 (6.5 µg/mL) for 16 h and LDLR protein expression was detected and quantified by (E) immunoblotting,(F) flow cytometry and (G) LDLR messenger RNA by qPCR (H) Immunofluorescence confirmed LDLR expression in human LEC. Representative scatterplot of red and green pixel intensities of cholera toxin subunit B (red) and anti-LDLR (green) in lymphatic endothelial cells and a reconstruction of colocalized voxels (scale bar, 20 µM). (I) Protein expression of VEGFR-3 was measured in human LEC by flow cytometry following a 16 h incubation with 6.5 µg/mL human recombinant PCSK9. n=3-9. Statistics: *p < 0.05, ***p < 0.001, ****p < 0.0001.