In situ hybridization In situ hybridization was carried out according to standard procedures

reated with siRNA for 3 days. Cells were lysed and the levels of GPC5 were examined by Western blotting with anti-GPC5 Rabbit mAb. Lane 1, control siRNA-treated cells; Lane 2, GPC5 siRNA-treated cells; Lane 3, markers. Growth curve. siRNA-treated cells were then seeded in 96-well culture plates and proliferation was assessed by CCK8 assay at the indicated time points. An exponential approximation line of control or siRNA-treated dots was drown between for 14 to 86hr. The results are expressed as the mean S.D. of quintuplicate wells. P < 0.05. Cell-cycle distribution of U3-DT cells. Cell cycle distribution of U3-DT cells incubated in medium containing siRNA or not is shown. Immunofluorescence profile of U3-DT cells. Double immunofluorescence labeling with anti-human GPC5 and Ptc1 antibodies in U3-DT cells. Nuclei are labeled with DAPI. Arrows: GPC5 and Ptc1 positivemerged spots are clearly visible in U3-DT cells. Scale bar = 50 m. doi:10.1371/journal.pone.0126562.g006 provided several important pieces of evidence regarding transformation-related changes at different stages of prolonged PubMed ID: culture. A summary of the alterations in cytological features and gene expression PubMed ID: over the course of the transformation process is shown in 16 / 23 Alteration in Gene Expression on Transformation Note:, and denote the up-regulated, down-regulated, or control-level expression of major genes shown in Fig 3B. F, fibroblastic-like; NT, not tested. doi:10.1371/journal.pone.0126562.t001 instability in chromosome 13 have a diploid karyotype during the first 90 PDL, and they preferentially lose one copy of chromosome 13 upon prolonged culture. The loss of a whole chromosome 13, even a single copy, causes significant damage to near-diploid cells, but some survive, subsequently, acquire additional aneuploidy and tumor-related gene expression. Finally, unstable triploidy purchase CP 868596 confers tumorigenicity and is a dominant feature of tumors. This karyotypic heterogeneity of genomes is consistent with the neoplastic progression theory, which holds that tumorigenesis occurs by a complex evolutionary process: according to this theory, progression develops as genetic instability is acquired, leading to the accumulation of genetic alterations and the continual selective outgrowth of variant subpopulations of tumor cells with a proliferative advantage. By contrast, despite having a normal diploid karyotype with no detectable chromosome abnormalities, hTERT-transduced adult human MSCs formed tumors after longterm culture; however, these cells exhibit deletion of the Ink4a/ARF locus and epigenetic silencing of DBCCR1. A similar phenomenon was reported in hTERT-immortalized human fibroblast cell lines, which have a diploid karyotype and in which Ink4a is deleted; however, these cell lines require active H-Ras or H-Ras and inactive p53 for tumor formation, resulting in the acquirement of chromosome abnormalities. Therefore, these reports suggest that h-TERT-mediated immortalization does not significantly affect genome integrity at the chromosome level, and that h-TERT-associated genetic and/or epigenetic alterations contribute to tumorigenicity rather than karyotype alterations. In addition, the genetic and/or epigenetic alterations may largely reflect differences in the pre-existing genetic makeup of each strain and the culture conditions. Secondly, we showed that UE6E7T-3 acquired properties of transformed cells even without RAS- and MYC-mediated transduction. However, this transformati