In vitro methylation assay of the bBoule promoter

ling of stained U3-DT cells was performed with an ImageXpress Micro. Immunocytochemistry Cells cultured on coverslips were washed in phosphate-buffered saline, fixed in 4% paraformaldehyde in PBS and then blocked with 1% BSA in PBS. The cells were stained with an anti-GPC5 antibody, or anti-Ptc1 antibody, and Alexa Fluor 488 or 594 labeled secondary antibody. The cells were then counterstained with DAPI and visualized using a fluorescence microscope. Results Phenotypic characteristics of UE6E7T-3 cell line This laboratory has previously demonstrated that aneuploidy, specifically the loss of one copy of chromosome 13, arises in the immortalized hMSC line UE6E7T-3 after prolonged culture. We wished to investigate whether continuing culture after the appearance of aneuploidy would induce transformation of UE6E7T-3. To this end, we analyzed the phenotypic characteristics of the cells at various PDL. We monitored the growth characteristics of UE6E7T-3 cell line during long-term culture. Alterations were observed in population doubling time. DT was 40 hours at initial stage and it slightly became longer at next stage, and decreased to 2822 hours afterward more than 150 PDL. Over the course of culture until PDL 252, UE6E7T-3 cells exhibited typical fibroblastic morphology with a uniform bipolar spindle shape, and no obvious morphological changes were observed. Typical markers associated with hMSCs showed similar patterns at Stage IIV, including markers that are expressed in hMSCs and those that are not, although expression of CD90 and CD105 was slightly reduced at later stages. In additionally, UE6E7T-3 cells can differentiate into adipocytes and osteocytes, an important characteristic of MSCs. Changes in karyotype and neoplastic transformation during prolong cultivation The growth rate at PDL 252 of 22 hours) was 2-fold higher than the rate at PDL 65. Distinct alterations in numerical and structural karyotypes appeared as PDL increased. As shown in Fig 2A, nearly 90% of the cell population contained 46 chromosomes at PDL 62. By PDL 92, this proportion had decreased markedly to 17%, and a new population that contained 4445 chromosomes had appeared. Between PDL 92 and PDL 147, cells were unstable and comprised several populations differing in karyotype: near-diploid, with 4445 chromosomes; diploid; near-tetraploid, with 8391 chromosomes; and several minor populations. These populations were gradually replaced by near-tetraploid cells with 7382 chromosomes 6 / 23 Alteration in Gene Expression on Transformation Fig 1. Phenotypic characteristics of UE6E7T-3 during long-term in vitro culture. Growth curve. UE6E7T-3 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 cells were seeded at 5X103 cells / ml in POWEREDBY 10. When cells were subconfluently grown, the cells were passaged with trypsin as shown in the text. After counting cell numbers, aliquot of the cultured cells was cultured continuously. Phase-contrast images of UE6E7T-3 at four stages. Scale bar, 300 m. Flow cytometry. UE6E7T-3 cells were plotted. X-axis is fluorescence intensity. Y-axis is number of cell. doi:10.1371/journal.pone.XAV-939 web 0126562.g001 7 / 23 Alteration in Gene Expression on Transformation Fig 2. Phenotypic alterations of UE6E7T-3 during long-term culture. Changes in chromosomal number at various culture PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 stages. Chromosomes were counted by DAPI staining of 5080 metaphase spreads at each PDL. Distribution pattern of cells at PDL 62, 92, 118, and 147 were rearranged from raw data from a previous report. Multicolored fluorescence i