3 4 dhpaa

Restriction enzymes, DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase were purchased from Takara Bio, Inc. (Dalian, China). Hieff Clone^TM Plus Multi One Step Cloning Kit was purchased from YEASEN Biotechnology Co., Ltd. (Shanghai, China). Oligonucleotides were purchased from Life Technologies (Shanghai, China). 4-Hydroxyphenylacetate, tyrosine, tyrosol, hydroxytyrosol, tyramine, dopamine, 3,4-DHPAA, 4HPAA, sodium periodate, dansylcholoride, and NADH were all purchased from Sigma-Aldrich (St. Louis, USA).
Escherichia coli strain MC1061, BL21(DE3), and BW25113 were used for cloning, protein overexpression, and hydroxytyrosol biosynthesis, respectively. Strains were grown at 37 °C. Kanamycin (50 μg/mL) were supplemented when necessary. Hydroxytyrosol biosynthesis was performed in yeast extract M9Y medium (M9 minimal salts (Becton, Dickinson and Company), 1% (w/v) glucose, 5 mM MgSO₄, 0.1 mM CaCl₂ supplemented with 0.025% (w/v) of yeast extract)^{14 (link)}, while other cultures were done in Luria-Bertani (LB) medium if not specially indicated.

THP-1 (ATCC TIB202) cells, an in vitro model extensively used to study human monocytes and macrophages [48 (link)], were maintained in RPMI media (Cat# 11875093, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cat# 10437028, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C in a humidified incubator with 5% CO₂. The cells were subcultured every 2 to 3 days in order to maintain a cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL. THP-1 cells were seeded in assay plates in RMPI plus 10% FBS medium containing 25 ng of PMA (Cat# P8139, Sigma-Aldrich, St. Louis, MO, USA) per mL in order to induce differentiation of the THP-1 cells. After 24 h of cell plating, macrophages were activated with 5 pg/mL LPS (Cat# L8274, Sigma-Aldrich) and 10 ng/mL INFγ (Cat# SRP3058, Merck, Kenilworth, NJ, USA), and treated with 10 μM hemin (Cat# 51280, Sigma-Aldrich), 10 μM 3,4DHPAA (Cat# 850217, Sigma-Aldrich), or 10 μM 4HPAA (Cat# H50004, Sigma-Aldrich) for 72 h (Scheme 2).

Dulbecco’s cell culture medium, bovine serum albumin and 3,4-DHPAA, p-CoA, VA and FA were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Fetal bovine serum was purchased from Laborclin (Campinas, São Paulo, Brazil). Tissue culture flasks and cell scrapers were purchased from Nunc (Roskilde, Denmark). All the chemicals were of analytical grade.