4.5 g l glucose

DS and HEP/UFH from porcine mucosa and 4-sulfated (CS-4S) and 6-sulfated (CS-6S) CSs from porcine or shark cartilage, employed as GAG’s standards, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 6th international Heparin Standard (2154 UIs per vial, Lot No. 07/328) was obtained from the National Institute for Biological Standards and Control (Potters Bar, UK). The FucCS from Holothuria grisea was kindly provided by Dr. Gustavo Santos (Federal University of Rio de Janeiro, Rio de Janeiro, Brazil). Human colon carcinoma cells (LS180) purchased from ATCC (Manassas, VA, USA) were grown in minimum essential medium-α (Invitrogen; Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen). Mouse colon carcinoma cells (MC-38) expressing green fluorescent protein (MC-38GFP) [8 (link)], provided by Dr. Lubor Borsig, were grown in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen). All reagents were purchased from Sigma-Aldrich unless otherwise stated.

A previously published cell
line with overexpressed 17β-HSD10
(HEK-293-HSD10)^{15 (link)} was used to measure 17β-HSD10
inhibition in the cellular environment. For this purpose, the (−)–CHANA
fluorogenic probe was used. The cells (density 1 × 10⁴ cells per well) were seeded in DMEM (200 μL) without phenol
red (Gibco). The cells were supplemented with fetal bovine serum (10%,
Gibco), l-glutamine (2 mM), nonessential amino acid additives
(Gibco), and 4.5 g/L glucose (Sigma) into black clear bottom 96 well
plates (Brand, 781971). The cells were incubated for 20 h following
the treatment with compound 23 in DMSO/DMSO only (vehicle
control). After 2 h of compound treatment, the (-)-CHANA probe was
added at the final concentration of 20 μM. The changes in fluorescent
intensities were measured immediately after (-)-CHANA addition and
2 h later. The fluorescence intensities of the CHANK product were
taken by using the TECAN SPARK 10 M instrument (Ex/Em = 380/525 nm).
The residual 17β-HSD10 activity was calculated as ΔF between
2 and 0 h after (-)-CHANA treatment, and the data were normalized
between nontreated HEK-293-HSD10 and native, nontransfected HEK-293
controls (using relative response ratio). Compound 23 was used at 1, 5, 10, 15, and 25 μM concentrations to detect
the ability to penetrate the cells and to influence the 17β-HSD10
enzyme activity inside the cells.

Hek-Blue hDectin-1b cells (InvivoGen) were grown in DMEM medium with 4.5 g/L glucose (Sigma-Aldrich), 10% heat inactivated Fetal Bovine Serum Premium (FBS) (Biowest), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Sigma-Aldrich), 100 μg/mL Normocin™ and 1 µg/mL puromycin (both from InvivoGen) in vented T75 flasks. When cells reached 80% confluency, they were re-seeded at 5 × 10⁴ cells/well in flat-bottom 96 well-culture plates and stimulated with 10, 50 and 100 µg/mL of WGP^®-Soluble, WGP^®-Dispersible, or Zymosan in HEK-Blue™ Detection medium for 16 h. Substrate hydrolysis by secreted alkaline phosphatase (SEAP), upon activation of the receptor, was assessed at 620-655 nm according to manufacturer’s instructions in a BioteK™ µQuant Microplate Reader using Biotek™ Gen5™ Data Collection and Analysis Software (Thermo Fisher Scientific, Waltham, MA, USA).