Fig. 5: LRP5 transports PUFAs to intracellular compartments via internalization.

a, b Neutrophils from WT and neutrophil-specific LRP5 KO mice on the essential fatty acid-free diet and cultured with or without EPA (100 µM) for 3 h were stained with the anti-PUFA and anti-TGN38. Quantification of anti-PUFA staining intensity in Panels b and c with normalization against TGN staining intensity is shown in (b) (n = 6 cells per group from two independent experiments). c, d Flow cytometryt imaging analysis of neutrophils from WT and neutrophil-specific LRP5 KO mice on essential fatty acid-free diet and cultured with EPA, followed by staining with anti-PUFA and anti-TGN38. Quantification of anti-PUFA staining intensity after normalization against anti-TGN38 staining intensity is shown in (d) (n = 5969 cells for WT and n = 7014 cells for KO). e Neutrophils from WT and neutrophil-specific LRP5 KO mice on the essential fatty acid-free diet were cultured with EPA and subjected to proximity ligation assay (PLA) using antibodies for PUFA and LAMP1 (PLA fluorescence signal, magenta) and counterstained with DAPI (blue) (n = 32 cells for WT and n = 30 cells for KO from three independent experiments). f LRP5 KO HEK293T cells were transfected with the LRP5 or LRP5 ΔLDL expression plasmid and treated with or without EPA. Cell surface LRP5 proteins were detected by Western blotting after the precipitation of biotinylated cell surface proteins using avidin-columns (n = 3 per group from three independent experiments). PM plasma membrane. g LRP5 KO HEK293T cells with re-expressed WT LRP5 were cultured in the charcoal-filtered medium overnight, followed by incubation with EPA for the indicated period with or without dynasore (80 µM) treatment. The co-localization of EPA and LRP5 was detected using PLA (n = 10 cells for 0 min and 5 min without Dynasore, n = 13 cells for 5 min with Dynasore). Data in this figure are all presented as mean ± sem with p values (Student’s t-test, Two-tailed, unpaired).