Desmosterol

NBD-labeled cholesterol was obtained from Invitrogen (Carlsbad, CA, USA). N-Methyl-N-trimethylsilyl-trifluoracetamide, lathosterol, desmosterol, 25-HC, compactin, mevalonate, methyl-β-cyclodextrin, and water-soluble cholesterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ezetimibe was purchased from Selleck Chemicals (Houston, TX, USA). Lipoprotein-deficient serum was obtained from Merck Millipore (Billerica, MA, USA). Caffeine and catechin standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Non-catechin flavonoid standards were obtained from Baoji Herbest Bio-Tech Co., Ltd (Beijing, China). The hawk tea leaves were purchased from Yuehua Tea Group Co., Ltd (Yaan, China). The green tea (Xihu Longjing) and black tea (Qimen black tea) leaves were purchased from Beijing Hanmojuxiang Trade Center (Beijing, China).

For cholesterol enrichment, both, cholesterol and desmosterol (both Sigma-Aldrich) dissolved in ethanol were continuously added to BUVEC cultures (n = 5) at 5 μM final concentration. Although not being physiological the administration of cholesterol at low concentration (in ethanol) according to Xu et al. [35 (link)] was chosen to reduce its toxicity in long-term application. Supplementation of cholesterol complexed to cyclodextrin was not applicable to long-term culture in our hands owing to its high cytotoxicity.
For LDL enrichment, LDL (Sigma-Aldrich, 10 mg/mL final concentration) was supplemented to E. bovis-infected BUVEC (n = 5) from 10 days pi onwards. To artificially enhance lipid droplet formation in host cells, oleic acid (Sigma-Aldrich) was supplemented in BSA formulation complexes [36 ] to the cell culture medium. Direct conjugation was performed by mixing oleic acid-free BSA (fraction V, Roth) with oleic acid at the molar ratio of 6:1 (oleic acid:BSA). BUVEC (n = 3) were treated with 5 μM oleic acid/BSA complexes for an induction period of 1 h, and thereafter continuously with 2.5 μM to prevent toxicity. Treatments were repeated every second day from 8 days pi onwards.

All reagents were of analytical grade or of the highest grade available. Antibiotic mixture of penicillin/streptomycin (10,000 U/mL/10,000 mg/mL), fungizone (250 mg/mL), and heat-inactivated fetal bovine serum (FBS) were obtained from GIBCO Invitrogen (Barcelona, Spain). Collagen G was obtained from Merck (Darmstadt, Germany). 4-Fluorobenzaldehyde (≥98%), collagenase from Clostridium histolyticum Type IA, desmosterol (≥84%), dexamethasone, ethylene glycol-bis-(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA), gentamicin, insulin solution from bovine pancreas (10 mg/mL), methoxyamine hydrochloride (≥98%), N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS), O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA, ≥99%), sodium chloride (NaCl, ≥99.5%), thiazolyl blue tetrazolium bromide (MTT, ≥98%), thymol (≥98.5%), Triton X-100, trypan blue solution, Williams’ E medium, and all standards used throughout the work were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Methanol (≥99.9%) and pyridine (≥99%) were purchased from VWR (Leuven, Belgium).