Dii oxldl

To assess ox-LDL binding to the cell surface, THP-1 cells with WT CAP1 overexpressed or knocked down using siRNA were pre-incubated with recombinant human PCSK9 (2 μg/mL) for 30 min. Subsequently, 10 μg/mL of DiI-ox-LDL (Thermo Fisher Scientific; L34358) was added for 1 h and the cells were then fixed with 1% formaldehyde and washed with cold PBS. Following centrifugation, the cells were washed with FACS buffer (PBS supplemented with 0.5% BSA) and analyzed using a FACSCanto II Flow Cytometry System (BD Biosciences).
To assess VLA-4 activation, THP-1 cells with CTRL or CAP1 siRNA were pre-incubated with PBS or recombinant human PCSK9 (2 μg/mL) for 30 min. Subsequently, the cells were washed twice with cold PBS, dispersed by passing through a 40-µm strainer (Falcon Cell Strainer, Corning), and resuspended in FACS buffer. After incubation with the FITC-tagged VLA-4 antibody (Merck Millipore, Burlington, MA, USA Merck; FCMAB389F) at 4 °C for 1 h, the cells were washed with cold PBS, and FACS analysis was performed. Flow cytometry analysis and sorting (BD FACSCanto II, LSR II, and FACS Aria III,; Franklin Lakes, NJ, USABD Biosciences) were performed using several antibodies specific for VLA-4, CD11b (BD Biosciences, 553310), and F4/80 (eE-Bioscience, San Diego, CA, USA; 17-4801-82). Data were analyzed using FlowJo version 10.0.5.

C‐MSC were cultured for 72 h in AM with 5 μM GW9662 (Sigma‐Aldrich, St. Louis, Missouri, USA). The treatment was added to the medium every 8 h. To check OxLDL internalization, 10 μg/ml DiI‐OxLDL (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added 3 h before the end of the experiment, following the protocol described above (see “OxLDL internalization assay”). Pictures were acquired with Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each biological sample, 15 fields were examined.

Ginkgetin and GSK1016790A (GSK101) were purchased from Sigma-Aldrich (St. Louis, MO), and FLIPR calcium 6 assay kit was obtained from Molecular Device (Sunnyvale, CA). Human native LDL (nLDL) was purchased from Stemcell Technologies (Vancouver, Canada). We incubated nLDL with 5 µM CuSO₄ for 12 h at 37 °C to prepare copper-oxidized LDL (oxLDL)^{7 (link),14 (link),16} . Fluorescent dye-labeled, DiI-oxLDL, was purchased from Invitrogen (Carlsbad, CA), and MCSF from R&D (Minneapolis, MN). Antibodies for FACS analysis were: TRPV4 (Millipore; Burlington, MA), CD36 (R&D; Minneapolis, MN), TLR4 and TLR6 (Novus Biologicals; Centennial, CO). PE-conjugated secondary antibodies were from R&D (Minneapolis, MN), and PE-conjugated isotype controls were from BD (Franklin Lake, NJ). For quantitative polymerase chain reaction (qRT-PCR), we used RNeasy kit from QIAGEN (Redwood City, CA). All primers were purchased from Bio-Rad (Hercules, CA). Cell culture media, cell culture related products, and western blot reagents were obtained from Thermo Fischer Scientific (Waltham, MA).

The assay was performed using an oxidized LDL (oxLDL) uptake assay kit (601180, Cayman) or DiI-oxLDL (L34358, Invitrogen). The BMDMs from Apoe−/− mice were incubated with oxLDL-DyLight 488 or Dil-oxLDL (10 μg/mL) for 4 h. After unbound LDL was washed out, the cells were fixed with 4% paraformaldehyde. The fluorescent cells were observed using an Eclipse Ti2 microscope (Nikon, Japan).

PMs and HASMCs were incubated in medium containing 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI-oxLDL, Invitrogen) to assay their ability of cholesterol uptake. PMs and HASMCs (1.0×10⁶ cells/well) in 12-well plates were incubated with 10 μg/mL DiI-oxLDL in presence or absence of formononetin for 6 h at 37°C in CO₂ incubator. Fluorescent images were captured with microscope and then fluorescence intensity was measured by ImageJ.