Dulbecco modified eagle medium (dmem)

U87MG human malignant glioma, SK-MEL-28 human melanoma, SK-OV-3 human ovarian cancer, and A549 human lung cancer cell lines were purchased from ATCC. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Biosera, Nuaille, France), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Biosera), and with 1% Penicillin/Streptomycin (Biosera). Cells were cultured in sterile T75 flasks with ventilation cap (Sarstedt, Nümbrecht, Germany) at 37 °C in a humidified atmosphere with 5% CO₂ in ESCO CelCulture Incubator (ESCO, Friedberg, Germany). Manipulations with the cells were performed in biosafety cabinet (laminar) ESCO Sentinel Gold class II model AC2-4E8 (ESCO).

Murine mammary adenocarcinoma 4T1.2 cells [24 (link)] were a gift from Robin Anderson (Peter MacCallum Cancer Centre, Melbourne, Australia) and were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Biosera, UK), 10% (v/v) foetal calf serum (FBS) (Biosera, UK) at 37 °C in 5% (v/v) CO₂ in air. 4T1.2luc cells were transduced with lentivirus expressing luc2 under the control of human ubiquitin C promoter (Caliper, MA, USA). Cells were screened monthly for Mycoplasma contamination.

SEMFs were isolated from colonic biopsies, as previously described [14 (link)]. Briefly, the collected colonic biopsies were washed and de-epithelialized using dithiothreitol 1 mM (DTT; Sigma-Aldrich, Darmstadt, Germany) and ethylene-diaminetetraacetic acid 1 mM (EDTA; Sigma-Aldrich, Darmstadt, Germany). Tissue denuded of epithelial cells was then transferred and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Biosera, Nuaille, France) plus 10% fetal bovine serum (FBS; Biosera, Nuaille, France) in 5% CO₂ at 37 °C. During culture, numerous nonadherent and adherent cells appeared in the culture flasks, and their culture medium was changed every 72 h. Denuded mucosal tissue was maintained in culture for up to 4 weeks, until numerous foci of myofibroblasts adhered on the flask surface. Tissue specimens were then removed, and intestinal myofibroblasts were characterized using immunofluorescence microscopy as being α-smooth muscle actin (α-SMA)- and vimentin-positive and desmin-negative, as shown in Figure 1.
Colonic subepithelial myofibroblasts at passages 2–5 were used in these studies. All experiments were performed with FBS-free media at 95% culture confluence with a stable ratio of supernatant volume-to-surface available for cell adhesion (1.5 mL:9.6 cm²). Colonic subepithelial myofibroblasts were passaged at a ratio of 1:3.

HeLa cells were routinely cultured in DMEM (Biosera, Nuaille, France) containing 10% v/v fetal bovine serum (Gibco, Waltham, MA, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Biosera, Nuaille, France) and 2 mM glutamine (Biosera, Nuaille, France) and maintained at 37 °C in a humidified incubator with 5% v/v CO₂/ 95% v/v air. Cells were seeded in 96-well plates (Corning, New York, NY, USA) at a density of 8000 cells per well and the next day were transfected with 50 ng DNA using the JetPRIME reagent according to the manufacturer’s instructions (Polyplus, Illkirch-Graffenstaden, France). In all transfections, 5 ng of the pFLuc plasmid used for normalization was also added. Then, 24 h after transfection, the EOs or their constituents were diluted in DMEM at a specific dilution, and 10 μL was dispensed into each well. Cells were incubated for 48 h, and then the supernatant (the cell medium) was collected for GLuc assays, while the cells were lysed for FLuc assays. GLuc and FLuc assays were performed as described below.

The MCF-7 cell line was obtained from the National Cell Bank of Iran (Pasteur
Institute, Tehran, Iran). The cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, Biosera, France) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin
(100 μg/mL). They were maintained in 5% CO₂ atmosphere at 37˚C. Cells were
cultured in 96-well culture plates at initial seeding number of 5×10³ cells per
well.

Human osteoblast-like MG-63 cell line (ECACC 86051601, Sigma Aldrich, St. Louis, MO, USA), obtained from an osteosarcoma of a 14 year old male, was cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM, Biosera Europe, Nuaille, France) supplemented with 10% (v/v) fetal bovine serum (FBS, Biosera Europe, Nuaille, France), 100 U/mL penicillin, 100 mg/mL streptomycin (PAA Laboratories GmbH, Austria), and 2.5 mM stable glutamine (Diagnovum GmbH, Ebsdorfergrund, Germany), at 37 °C under 5% CO₂ in a humidified incubator. Culture medium was refreshed as needed [36 (link)].

Human ESCC (KYSE‐30) and embryonic kidney (HEK293T) cell lines were purchased from the Pasteur Institute Cell Bank of Iran (http://en.pasteur.ac.ir/) and grown in RPMI 1640 medium (Biosera) and Dulbecco's modified Eagle's medium (DMEM; Biosera), respectively. Both culture media were supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Gibco, USA), 100 U/ml, and 100 μg/ml penicillin‐streptomycin (Gibco, USA) at a humidified atmosphere 37°C with 5% CO₂.