Psc833

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)).

The preparation of the plasmids for human α3(IV), α4(IV), and α5(IV) for nanoluciferase complementation system was previously described.^{13 (link)} In brief, the SmBiT and LgBiT fragments were fused to the C-terminal of α3(IV) and α5(IV) in the pFC36K SmBiT TK-neo Flexi and pFC34K LgBiT TK-Neo Flexi vectors (Promega), respectively. α4(IV) was subcloned into pEB multi-Hygromycin vector (Wako) and fused with 3×FLAG tag. Mutants of α3(IV) and α5(IV) were generated by site-directed mutagenesis as previously described.^{10 (link),13 (link)} The plasmid for PPIF/CypD/CypF (HG14689-CM) was from Sino Biological. For stable cell lines, α3(IV)-SmBiT, α4(IV)-3×FLAG, and α5(IV)-LgBiT wild type or G1244D were subcloned into pLVSIN vector (Takara), respectively, containing hygromycin, puromycin, or blasticidin resistance genes. The reagents CsA, PSC-833, and trimethyl amine N-oxide (TMAO) were from Sigma-Aldrich. Dimethyl sulfoxide, glucose, mannitol, sorbitol, sucrose, and taurine were from Nacalai Tesque. Trehalose was from Tokyo Chemical Industry. Brefeldin A (BFA) was from calbiochem. ALV (HY-12559) was obtained from Medchem Express. FK506/Tacrolimus was from Abcam.