Falcon flasks

Fibroblasts cell lines from 17 probands with PKS and nine normal controls were uniformly cultured in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 20% FBS (Hyclone South Logan, UT, USA), antibiotics (100 units/ml of penicillin and 100 ug/ml of streptomycin), and 1% L-glutamine (Life Technologies, Carlsbad, CA, USA). Approximately 9.0–15.0×10⁵ exponentially growing cells were seeded in 15 ml media in 75 ml falcon flasks (BD Biosciences, San Jose, CA, USA). Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. Cells were trypsinized (0.25% trypsin-EDTA) and split at 75–80% confluency. All 26-cell lines were cultured until they reached a population-doubling level between 15–35. Total RNA, and DNA were isolated from the same cultures for all the samples. Slides were also prepared for cytogenetic analyses from the same cultures. Standard G-banded karyotyping, single nucleotide polymorphism (SNP) array, and fluorescent in situ hybridization (FISH) analysis of 12p was performed on all cell lines at the start and end points of culturing to provide an accurate estimate of degree of mosaicism of these samples (Table 1).

The human glioblastoma cell line A172 was purchased from American Type Culture Collection (ATCC), and LBC3 cell line was developed from glioblastoma multiforme tissue taken from 56-year-old female patient subjected to surgical tumor resection, and was kindly given to us by Prof. Cezary Marcinkiewicz (Department of Neuroscience, Temple University, Philadelphia, PA, USA) [65 (link)]. Cells were cultured in high-glucose DMEM with 10% of heat-inactivated FBS Gold, penicillin (100 U/mL), streptomycin (100 μg/mL), and 2 mmol/L l-glutamine. Cells were cultured in Falcon flasks (BD Biosciences) in a 5% CO₂ incubator (Galaxy S+; Eppendorf, Hamburg, Germany) at 37 °C. Cells reaching subconfluence were detached from the culture plates using 0.05% trypsin 0.02%–EDTA in calcium-free PBS and counted in a Scepter cell counter (Merck Millipore, Billerica, MA, USA). Next, appropriate number of cells was seeded in DMEM 96- or 6-well plates. After 24 h of incubation, DMEM was removed and replaced with DMEM containing quercetin in the indicated concentrations. Control cells were treated with the vehicle-DMSO in the concentration of 0.25% in culture medium. Cells were then tested with the appropriate experimental protocols.

Human normal skin fibroblast cell line (CRL1474) was obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum GOLD (FBS GOLD), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were cultured in Falcon flasks (BD) in a 5 % CO2 incubator (Galaxy S+), at 37 °C. Subconfluent cultures were detached with 0.05 % trypsin, 0.02 % EDTA in calcium-free phosphate-buffered saline and counted in a Scepter cell counter (Millipore).

Human breast cancer cell line MDA-MB-231 and human skin fibroblasts (CRL1474) were obtained from American Type Culture Collection (ATCC). Cells were maintained in high-glucose DMEM supplemented with 10% heat-inactivated foetal bovine serum GOLD (FBS GOLD), 2 mM l-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were cultured in Falcon flasks (BD) in a 5 % CO₂ incubator (Galaxy S+; New Brunswick), at 37 °C. Subconfluent cultures were detached with 0.05 % trypsin and 0.02 % EDTA in calcium-free phosphate-buffered saline (PBS) and counted in a Scepter cell counter (Millipore).

The normal human knee articular chondrocytes (NHAC-kn) were purchased from Lonza (Walkersville, MD, USA). Cells were cultured in a specifically dedicated Chondrocyte Growth Medium (CGMTM) with the de-differentiation preventing supplement containing 0.2% R3-IGF-1, 0.5% rhFGF, 0.1% transferrin, 0.2% insulin, 5% fetal bovine serum (FBS), and 0.1% GA-1000 (gentamicin sulphate + amphotericin B), purchased from Lonza. Normoxic conditions were maintained by culturing cells in Falcon flasks (BD) in a 5% CO₂ incubator Galaxy S+ (RS Biotech, Irvine, UK), at 37 °C. Hypoxia was evoked by incubation of cells in an atmosphere containing reduced concentration of oxygen (1% O₂) in hypoxia chamber Galaxy 170R (Eppendorf Inc., Hamburg, Germany). Subconfluent cultures were detached from the flask surface with 0.05% trypsin 0.02% EDTA in calcium-free phosphate-buffered saline (PBS) and counted in cell counter Scepter (Millipore).

Human glioblastoma cell lines LN-229 and LN-18 were a kind gift of Prof. Cezary Marcinkiewicz from the Department of Neuroscience, Temple University, Philadelphia, USA. Cells were cultured in high-glucose DMEM with addition of 5 % of heat-inactivated fetal bovine serum GOLD (FBS GOLD), streptomycin (100 μg/mL), penicillin (100 U/mL), and 2 mmol/L L-glutamine. Cells were cultivated in Falcon flasks (BD) in a 5 % CO₂ incubator (Galaxy S+; New Brunswick) at the temperature of 37 °C. Cells reaching sub-confluency were detached from the culture dishes using 0.05 % trypsin 0.02 % EDTA in calcium-free phosphate-buffered saline (PBS) and counted in cell counter Scepter (Millipore). Belinostat was dissolved in dimethyl sulfoxide (DMSO) as 50 mmol/L stock solution and subsequently diluted in growth media keeping the final concentration of DMSO ≤1 % in culture.