Synthechol

THP-1 cells were seeded at a density of 2×10⁵ cells/well in 24 well plates and incubated in the presence of 120 ng/ml PMA (phorbol 12-myristate 13-acetate). After 24 h, fresh RPMI 1640 was added supplemented with 50 ng/ml human IL-4 (Sigma) or 50 ng/ml IL-4 and 4 μg/ml SyntheChol (Sigma). The medium was replaced every day with fresh medium containing the appropriate supplements. After three days the cells were fixed with methanol and stained with Giemsa (Sigma) and five randomly selected fields per well were acquired to determine MNGC formation. Two independent experiments were performed in triplicate (N = >7500 nuclei).

1×10⁶ RAW 264.7 macrophages were seeded into wells of a 6 well plate. Following overnight incubation the cells were treated with 10 mM MβCD for 1 h or with 4 μg/ml cholesterol (SyntheChol; Sigma) for 10 h. Untreated cells were used as control. The cell monolayers were washed once with PBS, dried and incubated in 1 ml isopropanol per well overnight at room temperature. The supernatant containing the extracted lipids was dried under vacuum and resuspended in reaction buffer provided with the Amplex Red Kit (Invitrogen). Cholesterol levels were determined fluorimetrically utilizing the Amplex Red Kit (Invitrogen) according to the manufacturer`s instructions and normalized to macrophage protein content. The remaining cell monolayers were solubilized in 1.5 ml/well 0.1 M NaOH and the total protein content was measured using the BCA assay. Two independent experiments were performed in duplicate.

DHCR24^−/− MEFs were plated at 10⁵ cells per well in a 6-well plate and allowed to adhere overnight. MEFs were infected with either WT or Δstmp1 mutant bacteria for 1 h in 500 μL fibroblast medium supplemented with a serum-free growth kit, washed with PBS to remove extracellular bacteria, and then gently scraped into 3 mL of medium. For the day 0 sample, 1 mL of infected cells was lysed in sterile water for 5 min, and the released bacteria were diluted in ACCM-2 and plated onto 0.25% ACCM-2 agarose plates, while the remaining cells were replated in a 24-well plate (5 × 10³ cells per well) under different cholesterol (Synthechol; Sigma-Aldrich) conditions. The medium was changed daily to ensure constant cholesterol concentrations. At 6 dpi, the cells were either lysed in sterile water for 5 min and the released bacteria diluted in ACCM-2 and spotted onto 0.25% ACCM-2 agarose plates or processed for immunofluorescence in order to measure the CCV size, as described above. Each of the three experiments was performed in biological duplicate, and the bacteria were spotted in triplicate for the CFU assay. At least 30 CCVs were measured for each CCV size experiment.