Mrc 5

HeLa, MCF-7 and MDA-MB-231 cells were obtained from Sigma-Aldrich and maintained as reported previously [22 (link)]. MRC-5, PC-3, DU 145 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). PC-3 and DU 145 were maintained in RPMI medium (Sigma-Aldrich) whereas MRC-5 cells were maintained in DMEM medium (Sigma-Aldrich). All media were complemented with 10% Fetal bovine serum (FBS, Sigma-Aldrich) 2 mM L-glutamine, 0.01% streptomycin and 0.005% ampicillin (Sigma-Aldrich). Cells were cultured under standard conditions in a 37 °C incubator containing 5% CO₂ in 95% humidity.

TIG-3 (Health Science Research Resources Bank) and Lenti-X 293T cells (Clontech/Takara Bio) were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (GE Healthcare), antibiotic–antimycotic; 50 U/ml penicillin, 50 μg/ml streptomycin, and 125 μg/ml amphotericin B (Gibco). MRC-5 and IMR-90 cells (National Institutes of Biomedical Innovation, Health and Nutrition) were maintained in Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 10% FBS, antibiotic–antimycotic, 50 U/ml penicillin, 50 μg/ml streptomycin, and 125 μg/ml amphotericin B. The cells were cultured under 5% CO₂ at 37 °C. Under our culture conditions, TIG-3, MRC-5, and IMR-90 cells reached replicative senescence at approximately PDL 80, 75, and 58, respectively. TIG-3 cells at PDL 41–59 and 77–83 were used as young and senescent cells, respectively. MRC-5 cells at PDL 50–55 and 70–75 were used as young and senescent cells, respectively. IMR-90 cells at PDL 40–45 and 55–58 were used as young and senescent cells, respectively.

Micro-culture plates (96-well; NUNC, Sigma-Aldrich) were pre-coated with BSA (2 μg/ml), fibronectin, rTsMFas1 and rTsMFas2 protein (each 10 μg/ml) overnight at 4 °C. Wells were blocked with PBS containing 0.2% BSA at 37 °C for 1 h and washed 3 times with PBS. Normal human lung fibroblast (NHLF; Lonza, Basel, Switzerland) and human fetal lung fibroblast (MRC-5; Sigma-Aldrich) cells were cultured in DMEM containing 10% fetal bovine serum at 37 °C in a 5% CO₂ incubator. Trypsinized cells (3 × 10⁵/ml) were inoculated into each well (0.1 ml of cell suspension per well) and incubated at 37 °C under 5% CO₂ atmosphere for 30 min. Unattached cells were removed by washing well with PBS twice. Attached cells were incubated with reaction buffer (50 mM citrate buffer containing 3.75 mM ρ-nitrophenyl-N-acetyl β-D-glucosaminide and 0.25% Triton X-100, pH 5.0) at 37 °C for 1 h. Enzyme activity was blocked by adding 50 mM glycine buffer (pH 10.4) containing 5 mM EDTA. The number of cells was estimated by measuring absorbance value at 405 nm using a microtiter reader (SpectraMax Plus 384; Molecular Devices, Sunnyvale, CA, USA).

The EOC cell line SKOV3 was purchased from ATCC (Rockville, MD, USA). Fibroblast cell line MRC-5 was obtained from the cell bank of the Chinese Academy of Sciences. Mesothelial cell line HMrSV5 was obtained from Jennio Biological Technology (Guangzhou, China). All the cell lines were authenticated by their source organizations prior to purchase, routinely checked for mycoplasma contamination and used within 4 months after frozen aliquot recovery. Primary normal ovarian fibroblasts (NFs), ovarian CAFs and tumor cells were obtained from EOC patient tumor tissues following procedures as previously described [53 (link)]. SKOV3 and primary EOC cells were maintained in McCoy's 5A medium and MRC-5, HMrSV5, primary NFs and CAFs were cultured in DMEM/F-12 medium with 10 % FBS and 1% penicillin/streptomycin (Thermo Scientific), at 37°C in a 5% CO₂ and 80% humidity incubator. TGF-β1 (50 ng/ml) (Sigma, St. Louis, MO, USA) was added to MRC5 cultures for 7-10 days to obtain the transformed CAFs (MRC5-CAFs). SKOV3 cells had been stably transduced with CMV-Fluc-IRES-RFP lentiviral particles (GeneChem, shanghai, China) and designated as SKOV3-Luc previously, which were further used in animal living imaging experiment.

The human non-small cell lung cancer cell line A549 (ATCC, Manassas, VA, USA) was maintained in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Verviers, Belgium). The human fetal lung fibroblast cell line MRC-5 (Sigma-Aldrich, St. Louis, MO, USA) was cultured in Eagle’s Minimum Essential Medium (EMEM; Lonza, Verviers, Belgium). Media were supplemented with 10% fetal bovine serum (PAA, Pasching, Austria), 50 µg/mL gentamycin (Sigma-Aldrich, St. Louis, MO, USA), and 1% non-essential amino acids (only EMEM medium; Sigma-Aldrich, St. Louis, MO, USA). Cultures were carried out in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. After reaching about 80% confluence during the exponential growth, the cells were harvested with trypsin–EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) and subcultured on 12- or 6-well plates (BD Falcon, Franklin Lakes, NJ, USA) for further experiments.

The HT-29 human colon adenocarcinoma cells, MCF-7 human fibroblast breast cancer cells, A-549 human lung cancer cells and MRC-5 normal human fibroblast lung cells were purchased from the American Type Culture Collection (ATCC, Boulevard Manassas, VA, USA). The cells were cultured and maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) medium for HT-29, MCF-7 and A549 cells and Eagle’s Minimum Essential Medium (EMEM, Sigma) for the MRC-5 cells. They were supplemented with 10% fetal bovine serum, 100 μg/mL penicillin/streptomycin, and 100 μg/mL of amphotericin B and the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

The primary pleural mesothelial cells NP2 and its hTERT-transduced derivative NP2-hT⁺ were cultured in ACL growth medium supplemented with 10% FBS. The primary Meso-CAFs (Meso109F, Meso125F) and their hTERT-transduced derivatives (Meso109F-hT⁺, Meso125F-hT⁺) as well as the human mesothelioma cell lines (MSTO-211H, SPC212) were all kept in RPMI-1640 medium with 10% FBS. MSTO-211H was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and SPC212 was kindly provided by Prof. R. Stahel (University of Zurich, Zurich, Switzerland). The SV40-immortalized, non-malignant human pleural mesothelial cell line Met5A and both normal primary human lung fibroblasts, MRC-5 and Wi38, were also obtained from ATCC (Rockville, MD, USA) and cultivated in the respective growth medium according to the supplier’s protocol (Met5A in RPMI-1640, MRC-5 in Dulbecco’s Modified Eagle’s Medium (DMEM) (#D5648, Sigma-Aldrich, St. Louis, MO, USA), and Wi38 in Eagle’s minimal essential medium (MEM) (#M5650, Sigma-Aldrich, St. Louis, MO, USA), all supplemented with 10% FBS). The primary pleural mesothelial cells and the primary Meso-CAFs used for experiments were at fewer than 15 passages. All used cells were maintained in a humidified atmosphere (37 °C, 5% CO₂), regularly passaged by trypsinization, and routinely checked for Mycoplasma contamination.