Fetal calf serum (fcs)

The ability of the Lactobacillus strains to adhere to both intestinal (Caco-2) and urinary (Hu609) epithelial cells was determined. In this assay, the cells were grown in a suitable medium line, in the case of Caco-2, it was Dulbecco’s medium; for Hu 609, RPMI 1640 medium was used. Media were supplemented with 10% heat-inactivated fetal calf serum (FCS, Lonza, Walkersville, MS, USA), 2 mM ultraglutamine (Lonza), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Polfa Tarchomin, Warsaw, Poland). For the adhesion assay, all types of epithelial cells (1 × 10⁵ cells per well) were seeded into 24-well plates and grown for 24 h in a humidified incubator with 5% CO₂ at 37 °C in culture medium supplemented only with 10% fetal bovine serum (FCS, Lonza) and 2 mM ultraglutamine (Lonza). Afterwards, the cells were infected with 1-mL bacterial suspension (10⁸ CFU/mL in culture medium with 10% FBS + 2 mM ultraglutamine) (Lonza). After 3-h incubation (37 °C, 5% CO₂), the suspension was aspirated, and the cells were washed three times with PBS (pH 7.3) to remove free bacteria. To establish the number of adherent bacteria, a monolayer was lysed using 1% Triton X-100. After that, 1/10 dilution of the suspension was seeded on MRS plates and incubated for 24 h at 37 °C. The results are expressed as bacterial colony-forming units (CFU) recovered per milliliter.

TLR4 and MyD88 gene expression was assessed in epithelial ovarian cancer cells A2780 and IGROV-1, and cervical cancer cells KB-3-1, as well as their chemoresistant daughter cells A2780cis (cisplatin-resistant), IGROV-1CDDP [40] (link), [41] (link) (cisplatin & paclitaxel-resistant) and KB-8-5-11 [42] (link) (colchicine and paclitaxel-resistant). IGROV-1 and IGROVCDDP cells were grown in antibiotic and chemotherapy-free RPMI (Sigma #R8758) with 10% FCS (Lonza, Belgium). KB-3-1 and its colchicine-resistant variant KB-8-5-11 were grown in DMEM (Sigma #D5671), containing 1% penicillin-streptomycin, 2% L-glutamine and 1% sodium pyruvate with 10% FCS (Lonza). KB-8-5-11 cells were routinely grown with colchicine in the media, and drug was removed for 3 days prior to the start of all experiments. All cell lines were maintained in a humidified atmosphere with 5% CO₂ at 37°C. All cultures were tested routinely and were mycoplasma-free.
Cells (1.25×10⁶ cells/10 cm dish) were plated and allowed to attach and grow for 3 days to reach 70–80% confluence. The cells were then trypsinised, washed in 10 mL PBS, centrifuged and the supernatant removed. The cell pellets were stored at −80°C prior to analysis. Total RNA was prepared using a RNeasy Mini Kit (Qiagen, UK) and the mirVANA miRNA Isolation Kit (Applied Biosystems) and expression was assessed as above.

Lox-IMVI, Malme-3M, Sk-Mel-5, and Sk-Mel-28 were obtained from the Department of Developmental Therapeutics, National Cancer Institute (NCI). WM-115, WM-266-4 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) and the SK-Mel-2 cell line was obtained from the American Type Culture Collection (ATCC). Cell lines were maintained at 37 °C with 5% CO2 in RPMI-1640 medium (Sigma) with 10% FCS (BioWhittaker). DLKP-A is an adriamycin-resistant, P-gp over-expressing variant of DLKP, established in the National Institute for Cellular Biotechnology from a squamous cell lung carcinoma sample [28 (link)]. DLKP-Mitox is a mitoxantrone-resistant sub-variant of DLKP that over-expresses BCRP that was used as a positive control for BCRP expression also established in the National Institute for Cellular Biotechnology [29 (link)]. DLKP-A, DLKP-Mitox and DLKP cells were maintained in DMEM/Ham’s F12 1:1 medium (Sigma) with 5% FCS (BioWhittaker). Stock solutions of ABT-751 (10 mM) (Abbott), and elacridar (3.56 mM) (Sigma-Aldrich) were prepared in dimethyl sulfoxide (Sigma-Aldrich). Clinical formulations of docetaxel (11.6 mM) and paclitaxel (7.03 mM) were obtained from St. Vincent’s University Hospital.

Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines were obtained from the Department of Developmental Therapeutics, National Cancer Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell lines were obtained from the European Association Culture Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines were maintained at 37°C with 5% CO₂ in RPMI-1640 medium (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal calf serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 were maintained at 37°C with 5% CO₂ in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Life Technologies, Dublin, Ireland), 1 mM non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Stock solutions of fascaplysin (Merck Millipore, Watford, UK) (10 mM), PLX4032 (Sequoia Research Products Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) were prepared in dimethyl sulfoxide (DMSO) PD0332991 (provided by Pfizer, Peapack, NJ, USA) (10 mM) was prepared in ultrapure water.

Human ALK-rearranged NSCLC cell lines, H2228 (variant 3, E6;A20), DFCI032 and H3122 (variant 1, E13;A20), were grown in DMEM (Lonza) with 10% FCS (Lonza). 293T packaging cells, were cultured in DMEM (Lonza) with 10% FCS (Lonza). The H2228 TTA A5 (shALK) were generated as previously described and shRNA expression wad induced with 1μg/ml doxycycline-hyciclate (Sigma) [43 (link)]. BEAS-2B cells were purchased from ATCC (# CRL9609) and grown in bronchiolar epithelial cell basal medium (Lonza; #CC-3170).
For ALK inhibitors, NVP-TAE684 was purchased from Axon Medchem and Crizotinib (PF-02341066) was kindly gifted by Pfizer.