Journal: Nature Communications

Article Title: PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor

doi: 10.1038/s41467-024-46336-2

Figure Lengend Snippet: a Fluorescence-activated cell sorting analysis of THP-1 cells demonstrating that treatment of PCSK9 for 1 h changed CAP1 localization to the membrane surface of monocytes in a dose-dependent manner (0, 500, 1000, and 2000 ng/mL) ( N = 3). b Immunofluorescent staining of THP-1 cells with CAP1 (green) and PCSK9 (red) demonstrating their colocalization (left panel) mainly to the membrane surface of monocytes (yellow). The colocalization between PCSK9 and CAP1 further increased 60 min after treatment with 2 µg/mL rhPCSK9 (right panel). Colocalization analysis within the membrane was performed using orthogonal views from different planes of confocal microscope images. Scale bar represents 5 μm. c A direct binding assay using the BLItz system showed that the binding affinity to PCSK9 was strongest for CAP1 (0.032 µM), intermediate for TLR4 (0.037 µM), and weakest for LOX1 (2.833 µM). d Immunoprecipitation analysis of THP-1 cells demonstrating the interaction between PCSK9 and CAP1 in monocytes. e Duolink Proximity Ligation Assay for detecting the interaction between PCSK9 and CAP1 after treatment with CTRL siRNA, TLR4 siRNA, or LOX1 siRNA. The interaction between PCSK9 and CAP1 was quantified by counting the red dots. The Proximity Ligation Assay showed that the interaction between PCSK9 and CAP1 was not affected by the presence or absence of TLR4 or LOX1 ( N = 4). The scale bar represents 5 μm. f Duolink Proximity Ligation Assay for detecting the interaction between PCSK9 and TLR4 with CTRL or CAP1 siRNA. The extent of the interaction between PCSK9 and TLR4 was quantified by counting the red dots ( N = 3). The scale bar represents 5 μm. g Immunofluorescence staining of CAP1 (green) and PCSK9 (red) in the arteries of AdV-CTRL or AdV-PCSK9-treated Ldlr −/− mice under S-flow and D-flow. CAP1 and PCSK9 were colocalized in all groups. CAP1 and PCSK9 expression increased in the AdV-PCSK9 injection group compared with that in the AdV-CTRL group under D-flow. The scale bar represents 20 μm. The differences between the groups were compared using the unpaired t -test (two-tailed). All experiments are independently performed and all data are presented as mean values ± SD.

Article Snippet: Lipid uptake was observed under an inverted light microscope (magnification, ×100 and ×40, Nikon; ECLPSE Ci-L) and the accumulated lipid droplets in the cells were stained red.

Techniques: Fluorescence, FACS, Membrane, Staining, Microscopy, Binding Assay, Immunoprecipitation, Proximity Ligation Assay, Immunofluorescence, Expressing, Injection, Two Tailed Test