Na butyrate

Cells were seeded at the density of 25,000 cells/cm². Individual cell lines were collected and cell number determined by hemocytometric counting for three consecutive days. Cell numbers were normalized to day 0 values. The count has been repeated three times and evaluated by two different operators. Treatment with Na Butyrate: LOX-1_RNAi DLD-1 and scramble_RNAi DLD-1 were seeded at the density of 5,000 cells/cm² in a 96-well plate. After 24 hours Na Butyrate (Sigma-Aldrich S.r.l., Milan, Italy) was added at the concentration of 5mM for 24 and 48 hours. Cells cultured in the absence of Na Butyrate was used as control, and MTT assay was performed to determine cell viability. After removing the supernatant of each well and washing twice by PBS, 20 μl of MTT solution (5 mg/ml in PBS) and 100 μl of medium were added. After three hours, the resultant formazan crystals were dissolved in dimethyl sulfoxide (100 μl) and the absorbance intensity was measured by a microplate reader (Bio-RAD 680, USA) at 490 nm with a reference wavelength of 620 nm. All experiments were performed in triplicate.

The continuous cell lines were provided for accessioning to the BRC by the original or secondary investigators [10 ]. Cell lines were grown at 37°C in a humidified atmosphere of air containing 5% CO₂. The basic growth media (Life Technologies, Darmstadt, Germany) were supplemented with 10–20% fetal bovine serum (Sigma Aldrich, Taufkirchen, Germany). For growth factor-dependent cell lines, specific growth factors or conditioned media containing growth factors were added. No antibiotics were added to the cultures. All cell lines were free of mycoplasma and other bacterial, yeast and fungi contaminations as tested by PCR and microbiological growth assays [11 (link)]. The authenticity of the cell lines was determined by DNA typing [1 (link)]. For the stimulation or induction of the replication competence of XMLV, PCR-positive cell lines were treated for 3–5 days with 10^–7 M 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Aldrich) and 3 mM Na-butyrate (Sigma Aldrich) or for 3–5 days with 1.25 μg/μl 5´-azacytidine (Sigma Aldrich).