Atcc htb 37

The bacterial and fungal strains (S2 Table in S1 File) used in this study were clinical isolates obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), the Centers for Disease Control and Prevention (CDC) and Biodefense and Emerging Infections Research Resources Repository (BEI Resources) (Manassas, VA, USA). RPMI 1640 (Thermo Fisher Scientific, Waltham, MA), YPD broth, YPD agar, cation adjusted Mueller-Hinton broth, brain heart infusion broth, and lactobacilli MRS broth (Becton, Dickinson and Company, Franklin Lakes, NJ) were purchased from commercial vendors. Phosphate buffered saline was purchased from Fisher Scientific (Waltham, MA). Yeast extract, L-cysteine, vitamin K, 3-(N-Morpholino)propanesulfonic acid (MOPS) and hemin were obtained from Sigma-Aldrich (St. Louis, MO). Human colorectal adenocarcinoma epithelial cells (Caco-2) (ATCC HTB-37), and monkey kidney epithelial cells (Vero) (ATCC CCL-81-VHG) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

High-glucose Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 Medium, Hanks’ Balanced Salt Saline (HBSS), non-essential amino acids (NEAA), L-glutamine, Penicillin–Streptomycin mix, lipopolysaccharide (LPS), Diclofenac, dihydrotestosterone (DHT), staurosporine (STS) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). LNCaP androgen-sensitive human prostate adenocarcinoma cell line (ATCC^® CRL-1740™), Caco-2 human colorectal adenocarcinoma cells (ATCC^® HTB-37™) and THP-1 (ATCC^® TIB-202™) were purchased from ATCC (Manassas, VA, USA). CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) and Apo-ONE^® Homogeneous Caspase-3/7 Assay were purchased from Promega (Madison, WI, USA). Oxygen Radical Antioxidant Capacity (ORAC) Assay kit was purchased from Cell Biolabs (San Diego, CA, USA). Transwell^® inserts were purchased from Millipore (Burlington, MA, USA). Fetal bovine serum (FBS) was purchased from Euroclone (Milan, Italy). Interleukin 1β (IL-1β), Tumor Necrosis Factor α (TNF-α), PSA and DHT ELISA kit were purchased from R&D Systems, PeproTech (London, UK), Abcam (Cambridge, UK), and Cloud Clone (Katy, TX, USA), respectively.

All the chemicals, including diosgenin, and the reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise stated. The reagents for cell culture and bacterial culture were purchased from GE Healthcare Life Sciences (Marlborough, MA, USA) and BD Diagnostics (Sparks Glencoe, MD, USA), respectively. The ELISA reagents and standard were purchased from eBioscience, Inc. (San Diego, CA, USA), while 10% neutral buffered formalin was purchased from Leica Biosystems Richmond Inc. (Richmond, IL, USA). The Caco-2 cell line (ATCC^® HTB-37™) was maintained in DMEM (L-glutamine, high glucose; Corning Inc., New York, NY, USA) supplemented with 10% fetal bovine serum.

VAL and 4-HBA cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on colorectal adenocarcinoma cell line Caco-2 (ATCC^® HTB-37™, ATCC, Manassas, VA, USA). Compounds’ stock solutions were prepared in dimethyl sulfoxide (DMSO), diluted in complete medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1 mM sodium pyruvate) and filtered on 0.22 µm filters. The concentrations tested were 245, 100, 30, 10 µM for VAL and 36, 18, 3.5, 1 µM for 4-HBA. DMSO 0.1% (the highest concentration in the dilutions) was added as control. Briefly, 1 × 10⁴ Caco-2 cells/well were seeded in a 96-well plate and treated with serial dilutions of VAL and 4-HBA for 24 h. The MTT solution (0.5 mg/mL) was then added to each well and incubated for 3–4 h. Formazan crystals were solubilized with 100 µL/well of lysis buffer (8 mM HCl and 0.5% NP-40 in DMSO) and the absorbance measured at 575 nm in a microplate reader (Power Wave HT, Biotek, Bad Friedrichshall, Germany). The cell viability was calculated by the following formula: $\% Cell viability=\left(absorbance sample÷absorbance control\right)\times 100$
Three independent experiments were performed with four technical replicates for each condition.