Blt 1

ORS keratinocytes were seeded at 2 × 10⁴ cells per well in a 48 well plate and incubated overnight. Cells were treated with 5 µM T0901317 or DMSO vehicle control (0.05%) for 3 days. Cells were washed in Epilife prior to incubation with BODIPY-cholesterol (Avanti lipids, Alabama, USA) (25 µM complexed 1:10 with methyl-β-cyclodextrin (MβCD; Sigma-Aldrich) for 2 h in the dark. Cells were washed in Epilife and equilibrated for 24 h with Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, 2 µg/ml Sandoz 58–035 (Sigma-Aldrich). Cells were washed in a 10 mM HBSS-HEPES solution (Lonza) and efflux was initiated by adding cholesterol acceptors, 10 µg/ml Apolipoprotein A-I (ApoA1; Sigma-Aldrich) or 25 µg/ml high-density lipoprotein (HDL; Sigma-Aldrich) with 5 µM PSC833 (Sigma-Aldrich), 10 µM BLT-1 (Sigma-Aldrich) or DMSO vehicle control (0.18%) for 4 h. Background efflux was performed in the absence of cholesterol acceptors, with or without PSC833 or BLT-1. Media was removed and fluorescence measurements performed in black-walled 96 well plates (excitation 485/20 nm, emission 528/20 nm).
Time 0 monolayers were incubated with 1% cholic acid (Sigma-Aldrich) for 1 h at room temperature on a plate shaker. Cells were scraped and fluorescence measured (excitation 485/20 nm, emission 520/20 nm). Efflux calculations were performed, as previously described (Sankaranarayanan et al. 2011 (link)).

Recombinant human TRAIL was purchased from BioVision (Milpitas California, USA). Recombinant TNF-α was from R&D Systems (Minneapolis, MN, USA). Rabbit anti-SR-AI antibody was from Abcam (Cambridge, UK). Rabbit anti-TRAIL was from Novus Biologicals (Littleton, CO, USA). Rabbit antibodies against ERK, phospho-ERK, p38, phospho-p38, JNK and phospho-JNK were all from Cell Signaling Technology (Beverly, MA, USA). SB202190, JNK Inhibitor II, U0126, BLT-1, staurosporine and z-VAD-fmk were all from Merck Millipore (Darmstadt, Germany). Poly(I:C) was from InvivoGen (San Diego, CA, USA). Lipopolysaccharide (LPS) was from Sigma (St. Louis, MO, USA). DiI-labeled acetylated LDL (DiI-Ac-LDL) and oxidized LDL (ox-LDL) were from Yiyuan Biotechnologies (Guangzhou, China).

HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM containing 10% fetal calf serum. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The HepG2 cells were seeded at a density of 6 × 10⁵/well on 6-well plates, and were grown to 70–80% confluence for 24 h before adding BLT-1 (Merck-Millipore, Darmstadt, Germany). Prior to the experiment, cells were washed twice with phosphate-buffered saline (PBS). Cells were cultured in the above medium or serum-free medium with 1% bovine serum albumin (BSA), containing one or more additives, i.e., BLT-1, HDL, and cholesterol, at different concentrations as described in the figure legends. HDL was purchased from Prospect Biosystems (Newark, NJ, USA), and cholesterol was obtained from Sigma-Aldrich (St. Louis, MO, USA).