Bodipy fl ldl

Hepatocytes were exposed for 3 h to 5 µg/ml dil-LDL (Life Technologies) or 5 µg/ml of BODIPY-FL-LDL (Life Technologies) in HCM medium. For uptake measurement, hepatocytes were washed five times with PBS then dissociated for 40 min in Gentle Cell Dissociation Reagent. Cells were recovered in PBS and analyzed by flow cytometry using the FACS ARIA III (Becton Dickinson).

All the antibodies used in this study are listed in Supplementary Table 2. Alexa-Fluor-conjugated-Dextran, Lysotracker dyes, Lysosensor dyes, BODIPY FL LDL, Phalloidin, and DAPI, were purchased from Molecular Probes (Invitrogen). SiR-Lysosome Kit was purchased from Cytoskeleton, Inc. Self-Quenched BODIPY FL conjugate of BSA was purchased from BioVision. Polybrene, Puromycin, EBSS, Rapamycin, and Bafilomycin A1 were purchased from Sigma-Aldrich.

For measuring uptake of fatty acids or cholesterol, cells were incubated in PBS containing 0.5 mg/mL C1-BODIPY 500/510 C12 (ThermoFisher, D3823), or PBS containing 0.1 mg/mL BODIPY FL C16 (ThermoFisher, D3821) for 20 min at 37 °C. For measuring uptake of cholesterol, cells were incubated in PBS containing NBD Cholesterol (ThermoFisher, N1148) at a final concentration of 10 mM for 15 min at 37 °C. For measuring LDL uptake, cells were incubated in PBS containing 0.3% BSA and 20 mg/mL BODIPY FL LDL (ThermoFisher, L3483) for 30 min at 37 °C. For measuring OxLDL uptake, cells were incubated in PBS containing OxLDL-DyLightTM-488 (1:20 dilution, Oxidized LDL Uptake Assay Kit, Cayman Chemical, #601180), or PBS containing 50 mg/mL DiI-labeled human high oxidized low density lipoprotein (Kalenbiomed, Cat# 770262-9) for 30 min at 37 °C. After incubation, cells were washed with MACS buffer (PBS containing 2% FBS) for surface staining. For measuring lipid peroxidation, cells were incubated in PBS containing 2 mM BODIPY 581/591 C11 reagent (ThermoFisher, C10445) for 30 min at 37 °C before live dead and surface staining. CD36⁺ CAFs or CD36^kd CAFs were sorted prior to C11 lipid peroxidation assay to avoid interference of tumor cells in the assay. The reagents were listed in the Supplementary Table S5.

The amount of LDL uptake was measured as previously described [17 (link), 30 (link)]. Briefly, the cells were treated with vehicle or tanshinone IIA for 24 h, which was then replaced with fresh serum-free medium and incubated with 10 μg/mL BODIPY^®-FL-LDL (Thermo Fisher Scientific) at 37°C for 24 h. The cells were detached, washed and re-suspended in PBS and examined by flow cytometry. Data were acquired from 10,000 cells (counts). The LDL uptake data were expressed as the relative percentage of the geometric mean fluorescence intensity.

HDL and LDL labeled with 1,1’-Dioctadecyl-3,3,3,3’-tetramethylindocarbocyanine percholorate (DiI-HDL and DiI-LDL) were purchased from Alfa Aesar. Cells were washed with DMEM containing 0.5% fatty acid-free bovine serum albumin (Sigma) (medium A), and medium A containing 5μg DiI-HDL or DiI-LDL was added to each well. After incubation for 2 h at 37°C, cells were washed twice with PBS and observed with a FluoView FV1000 laser scanning confocal microscope. HDL uptake was determined by using HDL Uptake Assay Kit (Bio Vision) according to the manufacturer’s protocol. LDL uptake was determined after incubation with medium containing 10μg/ml BODIPY FL LDL (Thermo Fisher) without FCS for 24 h at 37°C by using PowerScanHT (DS Pharma Biomedical) according to the manufacturer’s protocol.