Compactin

Luciferase assays were done using a modified version of previously described protocols (Oeffner et al., 2009 (link); Zelenski et al., 1999 (link)). Briefly, cells were plated in DMEM:F12 supplemented with 5% lipoprotein‐deficient serum (Sigma‐Aldrich) and 20 μM sodium oleate (Sigma‐Aldrich) in a 96‐well plate at a density of 15,000 cells/well. On Day 1, cells were transfected with a 1:10 mixture of Renilla luciferase driven by a constitutive thymidine kinase promoter and firefly luciferase driven by a hamster HMG‐CoA synthase promoter containing a sterol regulatory element (SRE) (gift of Timothy Osborne, Addgene #60444). FuGENE HD (Promega) was used as the transfection reagent according to the manufacturer's protocol. Four hours posttransfection, the media was changed to sterol‐deficient media (DMEM:F12 with 5% lipoprotein deficient serum, 50 μM lithium mevalonate [Sigma‐Aldrich], and 50 μM compactin [Sigma‐Aldrich], or sterol‐enriched media with the addition of 10 μg/ml water‐soluble cholesterol [Sigma Aldrich] and 1 μg/ml 25‐hydroxycholesterol [Sigma‐Aldrich]). After 24 h, luciferase activity was measured using the Dual‐Luciferase Reporter Assay System (Promega) in a SpectraMax L microplate luminometer with reagent injectors (Molecular Devices).

HLE-B3 cells were obtained from ATCC (Cat# CRL-11421) and cultured in Eagle’s minimum essential medium (ATCC) containing 20% fetal bovine serum (HyClone), 50 units/mL penicillin G, and 50 μg/mL streptomycin. Lipid depletion in HLE-B3 cells was induced with the treatment of 5% lipoprotein depleted fetal bovine serum (Kalen Biomedical LLC), 10 μM compactin (Sigma–Aldrich), and 50 μM mevalonate (Sigma–Aldrich)^{12 (link)}. 100 μM MG-132 (Sigma–Aldrich) was treated in HLE-B3 to increase nuclear stabilization of mature SREBP2^{98 (link)}. All cell culture was performed under 5% CO₂ atmospheric oxygen, at 37 °C in a humidified incubator.

Pretreatment and treatment media were DMEM/F12 media supplemented with 5% (v/v) lipoprotein-deficient serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) and statin (compactin, 5 μM) and Mevalonate (50 μM) to deplete cellular cholesterol.
For the Amplex Red assay, after siRNA transfection, Huh7 cells were washed once with PBS and refreshed with DMEM/high glucose supplemented with 10% (v/v) lipoprotein-deprived serum prepared in-house from fetal calf serum.
Cycloheximide (Sigma), MG132 (Sigma), and compactin (Sigma) were dissolved in DMSO. Mevalonate (Sigma) was dissolved in ethanol. Cholesterol-cyclodextrin (Sigma) was dissolved in filtered MilliQ water. Appropriate vehicle controls were used. Concentration of drugs used and treatment time are indicated in the figure legends.