Mem media

Three human fibroblast cell lines were used in this study, IMR90, WI38, and HDF, were all purchased from ATCC. IMR90 and HDF cells were both cultured in RPMI 1640 (Hyclone SH30027.01) supplemented with 10% FBS (Hyclone SH30070.03), 1% penicillin-streptomycin (Hyclone SV30030), and 1% L-Glutamine (Hyclone SH30034.01). WI38 cells were cultured in MEM media (Gibco 10370–021) supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-Glutamine.
Three-epithelial cell lines, A549, H358, and HPL1D were used to seed onto fibroblast-derived matrix were purchased from ATCC. HPL1D cells are immortalized peripheral lung epithelial cells and were used to compare the cancerous phenotype of A549 and H358 cell lines to normal. However, HPL1D cells have been shown to form soft-agar colonies in our lab, suggesting they are transformed. All epithelial cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% Glutamine, and 1% penicillin-streptomycin.
To mimic hypoxia, cells were treated with 1mM cobalt chloride for 16 hours.

For normal human fibroblasts, cells were synchronized in G0/G1 phase by contact inhibition. After cells reached into confluency, we changed media and waited for 2 days before experiment to ensure contact inhibition. For cancer cells, which cannot be synchronized by contact inhibition, cell culture medium was changed into isoleucine depleted MEM-media (Gibco) supplemented with 5% three times dialyzed serum and 1% penicillin, streptomycin and Fungizone solution as previously described [46 (link),47 (link)]. Cells were incubated in this media for 24 h to synchronize them into G1 phase. Cell cycle synchronization was confirmed with FACSCalibur Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

A375 (ATCC, VA) and 451Lu human melanoma cell lines kindly provided by Dr. Meenhard Herlyn (Wistar Institute, PA) were cultured in DMEM and MEM media respectively (GIBCO, Carlsbad, CA), supplemented with 10% FBS (GIBCO) and 1% antibiotics (10,000 I.U.·mL⁻¹ penicillin and 10,000 µg·mL⁻¹ streptomycin, GIBCO), at 37°C with 5% CO₂ in a humid environment. For dose-dependent studies, biotransformed (BSE) and non-biotransformed (NBSE) soybean extracts were dissolved in dimethyl sulfoxide (DMSO). Cells (70% confluent) were treated with soybean extracts for 24 h at 37°C with specified doses in complete growth medium, and harvested for further studies.

Vero cells (CCL-81) were obtained from ATCC (Manassas, VA) and cultured according to ATCC’s instructions in 96 well microtiter plates (Nunc) at 2 × 10⁴ cells/well in MEM media (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco) at 37°C in 5% CO₂. The neutralization activity of antibodies was determined by co-incubation with 80 ng/mL (260 pM) of full-length TcdA, with antibody and toxin added simultaneously to the cells. After 72 h of incubation, the cell viability was quantified with the Cell Proliferation Assay Reagent WST-1 (Roche Diagnostics, Laval, Canada) according to the manufacturer’s instructions. Briefly, the media was replaced with 100 μL of MEM (without fetal bovine serum) containing 10% of WST-1, incubated for 40 min at 37°C in 5% CO₂ and finally the absorbance read at 450 nm. The neutralizing activity was calculated as % inhibition:
A450_test is the absorbance of cells incubated with TcdA and varying concentrations of antibodies;
A450_low is the absorbance of cells incubated only with TcdA (0% inhibition); and
A450_high is the absorbance of cells incubated only with media (100% inhibition).

Human MSCs (hMSCs) were isolated from adipose tissue biopsies, and then cultured, expanded, characterized, and cryopreserved by staff at the Mayo Clinic Human Cellular Therapy Laboratory as previously described in [87 (link),88 (link)]. The release criteria included culture sterility, mycoplasma testing, and cytogenetic analysis, as well as cellular phenotyping, including CD90+, CD105+. CD73+, HLA Class I+, CD14+, CD44+, HLA DR, and CD45-. Cells were cultured in advance MEM media (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with human platelet lysate (5%; Lawson, Mill Creek, WA, USA), 100 U/mL Antibiotic-Antimycotic (Gibco Thermo Fisher Scientific, USA), 100 units/mL Glutamax (Gibco Thermo Fisher Scientific, USA), and 1000 USP units heparin (Novaplus, Lake Zurich, IL, USA) at 5% CO₂ and 37 °C.

BC cell lines RT4, HT1376, T24 (American Type Culture Collection (ATCC)), FL3 (Prof. Dan Theodorescu, University of Colorado Cancer Center, Aurora, CO, USA) and immortalized human bladder epithelium HCV29 (Prof. Jesper Zeuthen, Danish Cancer Society, Copenhagen, Denmark)) cells were used in the present study. T24 and RT4 cells were grown in McCoy’s 5 A media (Gibco, Life Technologies, Carlsbad, CA, USA). FL3 cells were grown in DMEM/F12 media (Gibco, Life Technologies) with 2% L-glutamine (Gibco, Life Technologies) and HCV29 cells were grown in DMEM (Gibco, Life Technologies). Finally, HT1376 cells were propagated in MEM media (Gibco, Life Technologies) with 1% non-essential amino acids (Gibco, Life Technologies) and 2% L-glutamine (Gibco, Life Technologies). The culture media was supplemented with 10% Fetal Calf Serum (FCS) (Gibco, Life Technologies) and 1% Penicillin-Streptomycin (Gibco, Life Technologies). Cells were cultured at 37 °C in an atmosphere of 5% CO₂. Cell line authentication was confirmed using Cell-ID (Promega, Fitchburg, WI, USA).

Organotypic cerebellum brain slice cultures were prepared according to previously published protocols [35 (link),36 (link),37 (link),38 (link)]. In brief, cerebella were derived from P10–P12 mice and separated from the rest of the brain. The cerebellum was dissected in ice-cold minimum essential media (MEM) supplemented with Glutamax (Gibco) until taken for cutting sagittal slices of 300 µm thickness using a McIlwain Tissue chopper. The separated slices were transferred onto humidified sterile culture membrane inserts with pore sizes of 0.4 µm (Merck Millipore, Watford, UK). Tissue slices on membrane inserts were cultured on a liquid layer of medium containing 50% MEM media (Gibco), 25% HS, 25% Earle’s Balanced Salt Solution (EBSS) (Gibco) supplemented with 130 mM D-glucose (Sigma) and 1% P/S. The cultures were first maintained at 37 °C in 5% CO₂ for 24 h prior to being used for in vitro experiments. After 24 h, the medium bathing the tissue was replaced with serum-free medium, into which treatments were added including vitamin K1 (100 µM) or warfarin (25 µM). After 72 h incubation, the culture medium was analysed for released Gas6 protein by ELISA assays for both total mouse Gas6 and Gla-Gas6 (below).