Gsk 264220a

Heparin, Heparinase, orlistat, and entecavir (ETV) were purchased from R&D Systems (Minneapolis, MN). The LIPG inhibitor GSK-264220A was purchased from Tocris Bioscience (Minneapolis, MN). Myrcludex B was purchased from Cosmo Bio (Tokyo, Japan). Primary antibodies to DYKDDDDK (FLAG) (#14793), ACTB (#4970), and GAPDH (#5147) were from Cell Signaling Technology (Danvers, MA); LIPG (ab24447) was from Abcam (Cambridge, UK); GFP (A-11122) was from Thermo Fisher Scientific; HBV core (HBP-023-9) was from Austral Biologicals (San Ramon, CA); and HBV preS1 was from GenScript (Tokyo, Japan). ELISA kit for LIPG (MBS2501814) was purchased from MyBioSource (San Diego, CA).

Cells were pre-incubated with chemical inhibitors (30 μM of Lalistat 2, 0.2 μM of GSK264220A, 200 μM of Genistein, 10 μg/ml of chlorpromazine, 5 μM of U18666A) for 1 hour and then continuously treated with lipid emulsions for 6 hours.
For the heparin assay, cells were treated with 50 UI/ml of heparin for 2 hours, followed by 2 times washing with PBS. Emulsions were subsequently added to the cells together with the same concentration of heparin.
Human LPL F:1 (Santa Cruz Biotechnology, California, USA) and 5D2 antibodies were added to the cell culture medium 2 hours prior to emulsion loading at the concentration of 2 μg/ml (1:100 dilution).
Genistein, Lalistat 2, chlorpromazine, and heparin were obtained from Sigma-Aldrich, Missouri, USA. GSK264220A was from Tocris (bio-techne), Abingdon, UK. LPL 5D2 antibody was contributed by Dr. Anne Beigneux, Department of Medicine, David Geffen School of Medicine, UCLA, USA.

Nile red staining was performed on cells grown on coverslips, fixed with paraformaldehyde, and incubated with Nile red (Sigma, 1mg/ml). Stained samples were mounted on slides and Nile red staining was evaluated under a fluorescent microscope (excitation 520 nm; emission 600 nm). Quantitation of lipid droplets was performed using ImageJ FIJI. For triglyceride measurements, the Triglyceride Colorimetric Assay Kit (Cayman Chemical) was used with minor modifications. Briefly, control and treated cells were collected and lysed, and then treated with the mixture of lipoprotein lipase, glycerol kinase, glycerol phosphate oxidase, and peroxidase with 4-aminoantipyridine and N-ethyl-N-(3-sulfopropyl)-m-anisidine. The resulting color was measured at 540 nm. To exclude free glycerol, a lipoprotein lipase inhibitor (GSK 264220A, Tocris) was used.