For the studies in this report, the sub-confluent passaged 10–87 VERO cells were designated as low-density passage 10–87 VERO cells (LD 10–87 VERO cells), while 10–87 VERO cells passaged at confluence were designated as high-density passaged 10–87 VERO cells (HD 10–87 VERO cells). The population doubling times (PDT) for LD 10–87 VERO cells and HD 10–87 VERO cells at p 250 were 26 h for the LD 10–87 VERO cells and 20 h for the HD 10–87 VERO cells. The LD and HD passaged cells used in this study and some of the tumors they formed were confirmed to be of simian origin by karyotyping and by PCR using

primers that recognize simian SINE sequences [28]. These cells have signaling pathway also been found to be free of 26 rodent

viruses and mycoplasma families (Radil, Columbia, MO). Adult (10 mice/dose) and newborn (NB) (3 litters/dose) athymic nude mice were inoculated subcutaneously (107 cells/mouse in 0.1 mL of PBS per cell line) above the scapulae. Tumors were documented to grow progressively by measurements in two dimensions until they were 15–20 mm in size, at which point the animals were euthanized. The tumor incidence in adult and newborn nude mice was recorded at weekly intervals over 12 month observation periods and plotted as survival curves. Wound-healing assays were performed as previously described [29] with some modifications. selleck chemicals Cells were plated 1 × 106 cells/plate in 60-mm culture dishes (Corning, Corning, NY) and allowed to form monolayers. When the cultures reached 90% confluence, they were serum starved for 8 h and the monolayers were wounded with a P200 micropipette

tip, washed with PBS and cultured in DMEM-10. Images of cell migration into the wounded areas were captured at 0, 3, 6, 9, 12 and 15 h. Total RNA from primary (p) African green monkey kidney (AGMK) cells and from cells from LD 10–87 VERO and HD 10–87 VERO cell banks established at every 10 passages from Sitaxentan p140 to p250 was extracted and purified using the miRNeasy mini kit according to the manufacturer (Qiagen Inc., Valencia, CA). The expression of signature miRNAs of these samples was measured using the TaqMan miRNA quantitative PCR assay [30]. Expression values were normalized to a small nucleolar RNA, RNU6 (Applied Biosystems). ΔCt values were calculated using the Ct values of the miRNA and the RNU6 for each corresponding sample. ΔΔCt values are calculated using the ΔCt values of the pAGMK cells and the experimental cell lines for each miRNA. The fold change over pAGMK was calculated.