Amberlite xad16n resin

Chemical extraction was performed as in Lauritano et al.¹⁰ by using Amberlite XAD16N resin (20–60 mesh, Sigma-Aldrich). The final extracts were freeze-dried and stored at −20 °C until screening. Before performing the assays, extracts were first diluted at 1 mg/mL with MilliQ water and 2.5% DMSO. A triplicate of control plus the same concentration of DMSO used in test wells was used in all assays. The anti-inflammatory assay was performed as in Lind et al. (2013). Briefly, ~10⁶ human monocyte THP-1 cells/mL (ATCC^{(R) TIB-202TM}) supplemented with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, SigmaAldrich) were seeded in 96 well plates and incubated at 37 °C, 5% CO₂ for 48 h in RPMI-1640 medium (Biochrom;10%FBS). After 72 h, 80 μL fresh RPMI medium and 10 μL/well (tested concentration 100 μg/mL) of test extract were added. The test was performed in triplicate. After incubation for 1 h, all samples were incubated with 1 ng/mL lipopolysaccharide (LPS; final concentration) for another 6 h at 37 °C. Enzyme-linked immunosorbent Assay (ELISA) was used to test TNFα secretion as in Lauritano et al.¹⁰ .

Crude extracts were prepared by growing each isolate in 250 mL Erlenmeyer flasks containing 100 mL of ISP2 medium or marine broth (without the addition of cycloheximide and streptomycin) (Table S1). The flasks were incubated in a rotatory incubator (Model 210, Comecta SA, Barcelona, Spain) at 28 °C, 100 rpm, in the dark. Cultures were grown for 1–2 weeks, depending on their growth rate, after which 1.5 g of Amberlite^® XAD16N resin (Sigma-Aldrich, MO, USA) was added to the cultures and left to incubate for an additional week. Cultures were extracted twice with methanol/acetone 1:1 (v/v). The organic layer was dried in a rotary evaporator and the resulting extract was dissolved in dimethyl sulfoxide (DMSO, ≥99.9%, Sigma-Aldrich, MO, USA) to obtain stock solutions with final concentrations of 3.0 mg mL⁻¹ and 1.0 mg mL⁻¹, to be tested in the bioactivity assays.