25 cm2 cell culture flask

Differentiation of MBCs into ASCs in was initiated in bulk PBMC cultures based on a previously established protocol [5] (link), using a stimulation cocktail composed of pokeweed mitogen (PWM), S. aureus Cowan I protein A (SAC) and CpG. IL-10 was added to the stimulation mix since a previous study showed that this enhanced the efficiency of MBC into ASC differentiation by more than 9 fold [6] (link). Briefly, PBMCs were thawed for 30 sec in a 37°C water bath and cold RPMI medium was immediately added drop wise. After washing, the cells were re-suspended in RPMI containing 10% FCS, 100 U/ml penicillin/streptomycin, 100 mM HEPES, 50 mM 2-β-Mercaptoethanol and 2 mM L-Glutamine (all Invitrogen) and counted. 1×10⁶ cells/ml were added to 25 cm² cell culture flasks (Greiner). Culture medium was supplemented with 50 ng/ml PWM lectin derived from Phytolacca americana (Sigma-Aldrich), 1∶5000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), 2.5 µg/ml ODN 2006 (Type B CpG nucleotide-human TLR9 ligand; InvivoGen tlrl-2006) and 25 ng/ml recombinant human IL-10 (PeproTech) and incubated at 37°C, 5% CO₂ for 5 days.

K1, Nthy‐ori 3‐1, PC‐9, and SK‐Mes‐1 cells were seeded in 25 cm² cell culture flasks (Greiner Bio One, Kremsmuenster, Austria). After reaching confluency, cells were washed and harvested, and extracted membrane protein fraction was analyzed as described elsewhere (Veshnyakova et al., 2012a). Proteins were detected using specific antibodies against Cldn1‐9 or with anti‐β‐actin (Thermo Fisher Scientific), respectively, and visualized by luminescence imaging (Lumi‐Imager, Roche, NY, USA).

The human EC cell line HEC-1A (moderately differentiated grade 2 adenocarcinoma; HTB112, ATCC, Manassas, VA, USA) [40 (link), 41 (link)] was cultured in Eagle’s minimal essential medium (MEM, 5.5 mmol/L glucose, equivalent to 100 mg/dL; Sigma-Aldrich, Munich, Germany) supplemented with 10% (v/v) charcoal-stripped fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 1% (v/v) non-essential amino acids (Sigma-Aldrich), 100 μg/mL streptomycin and 100 U/mL penicillin G (Gibco) at 37°C and 5% CO₂ in a humidified atmosphere. Cells were subcultured by detachment with 0.25% (v/v) trypsin/ethylenediaminetetraacetic acid (EDTA; Gibco) once a week and the medium was changed every 2–3 days. Experiments were carried out in 25 cm² cell culture flasks (Greiner, Kremsmünster, Austria) with a seeding density of 1.0 × 10⁵ cells in 5 mL medium in duplicates. After seeding, cells were allowed to attach and grow for 24 h before treatment.
Cells were treated with either 0.5 mmol/L metformin or 100 ng/mL insulin in culture medium supplemented with 10 nmol/L ß-estradiol (E2; all reagents purchased from Sigma-Aldrich) for 7 days with medium changes and renewed treatments every 2 days. Control cells were treated with substance-free medium supplemented with 10 nmol/L E2.

The osteoblast-like MC3T3-E1 cells (in the following referred to as MC3T3) were obtained from the German collection of microorganisms and cell culture (DSMZ, Braunschweig, Germany). They were cultured in 25 cm² cell-culture flasks (Greiner bio-one, Frickenhausen, Germany) in alpha MEM (ord. No. F 0925) supplemented with 1% penicillin/streptomycin (stock solution: 100 U/ml penicillin/100 μg/ml streptomycin), and 10% fetal bovine serum (all purchased from Biochrom AG, Berlin, Germany). The incubator ensured 95% humidity in a 5% CO₂ atmosphere at 37°C. Cells grown to confluence after approx. seven days were Trypsinated (0.05% Trypsin with EDTA 0.02%, PAN Biotech GmbH, Aidenbach, Germany) and subcultured.

The established human tumor cell lines, SK-OV-3 and EFO-27 and OAW-42 were cultured in a humidified incubator (37 °C, 5% CO₂, 95% air) and maintained according to the recommended cell line-specific culturing conditions. Cells were transferred into 25 cm² cell culture flasks (Greiner Bio-One, Frickenhausen, Germany) until they reached 70% confluency. For hypoxia experiments, cells were placed in a hypoxic chamber (3% O₂; mentioned as hypoxia). For acidosis experiments, culture media were supplemented with 2-Hydroxypropionic acid (Carl Roth, Karsruhe, Germany 0.2%, pH 6.2). Cells were cultured in parallel experiments under normal conditions (used as control). All treatments lasted for 24 h. Triplicates were generated. Afterwards, cells and cell culture media were processed separately. Cells underwent direct lysis according to the RNA isolation protocol, whereas cell culture media underwent extensive centrifugation (4000 rpm for ten minutes) before further processing.

A549 cells obtained from American Type Culture Collection (ATCC, Manassas, Virginia) (CRM-CCL-185) were propagated in 25 cm² cell culture flasks (Greiner, Germany) in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma Aldrich, USA) supplemented with 5% of FBS (fetal bovine serum, Sigma Aldrich) and 1% of penicillin-streptomycin (Sigma Aldrich) under stringent aseptic conditions, at 37 °C, 5% CO₂ for 3–4 days. 3 passages were performed before seeding of cells for particular experiments to increase the strength of the cells. The cells harvested from the flask with 0.25% Trypsin-EDTA solution (Sigma Aldrich) were used for further experiments.

Both lipoma-derived stem cells (LDSCs) and adipose-derived stem cells (ADSCs) were isolated by enzymatic digestion of tissue samples, respectively. Tissue samples were washed, cut into small pieces and placed in 0.1% collagenase type I solution (StemCell Technologies, Vancouver, CO, Canada), in a water bath at 37 °C for 45 min. Tissue homogenates were then vortex and filtered and cell culture media was added to stop collagenase. Stromal vascular fraction (SVF) of cells was obtained by centrifugation for 15 min at 1500 rpm, collected from the bottom of the tube and seeded in 25 cm² cell culture flask (Greiner Bio One, Kremsmünster, Austria) in standard cell culture medium (DM) that contained Dulbecco’s Modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM stable glutamine and 1% antibiotic-antimycotic solution (all purchased from Capricorn Scientific, Ebsdorfergrund, Germany). Cells were washed and media was changed 16–18 h after isolation to remove non-attached cells. After reaching the 70–80% confluency, first cell passage was performed (P1), which enabled purification of mesenchymal stem cells. Cells were cultured in standard cell culture conditions which mean temperature of 37 °C and humidified atmosphere with the presence of 5% CO₂. Medium was changed every three days.