CD44 and CD133 cell surface markers can be significantly enriched when grown under sphere-forming conditions

e activity of Cdc2, which is followed by cell cycle arrest at G2/M phase. Cdc2 phosphorylation was detected with a specific antibody, Cdc2Y15P. Increased Cdc2 phosphorylation was detected in the asf1-33 mutant compared to the asf1+ strain, which suggests that the cell cycle is delayed or arrested and that the checkpoint might be activated in the asf1-33 mutant at 36uC. Monitoring DNA replication by BrdU incorporation The BrdU incorporation assay was performed as described previously, with some modification. Cells expressing thymidine kinase under the control of the nmt1 promoter were incubated in EMM2 for more than 12 h to induce thymidine kinase gene expression. BrdU was added to the media and the cells were incubated at 26uC or 36uC for 4 h. Cells were collected by centrifugation and fixed with ethanol for 10 min. Cells were resuspended in 1 ml of 3.5 M HCl and incubated for 10 min to denature the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22179956 DNA and were then washed with PBS. The cells were then suspended in PBS containing 5% BSA and incubated at room temperature for several hours. Anti-BrdU antibody was added to each sample and followed by incubation at room temperature for 12 h. Cells were washed three times with PBS containing 5% BSA, and Alexa fluor 488-conjugated anti-rabbit antibody was added. After incubation at room temperature for several hours, cells were washed three times with PBS containing 5% BSA. Role of Asf1 in Genome Stability Name chk1 t1 chk1 t2 chk1t chk1 t4 cds1 t1 cds1 t2 cds1 t3 cds1 t4 cds1-check chk1-check pFA6a-F pFA6a-R chk HR 42-14 cnt1 F cnt1 R imr1 F imr1 R dg F dg R dh F dh R act1 RT F act1 RT R ura4 RT F ura4 RT R Sequence Cttatcccagccagtacaac cgtcgacctgcagcgtacgaatttgtgaaacatctgtaagaac cgagctcgaattcatgatgtgcacatcttttgaaaggtcg gaagacatgttaatgtctgcag ttattcatgggacgggaacc cgtcgacctgcagcgtacgaactcgaagaattgacgtgttc cgagctcgaattcatcgatgtaaccactctgtccacgtac tttcagcgataggtttggcg atcgtaagcaaagttattattgg aatgaaaaggtgagtttaggag tcgtacgctgcaggtcgacg catcgatgaattcgagctcg gctaggatacagttctctcaca gtaaagtaacagatatgtttcgc gcgtttcttcggcgaaatgc tcgatattactaggtgagtag gctgaggctaagtatctgtt atatccaactgacggcatcg tcataaagcaacatgggtg gtcgtagatgtgacgtcaac ggaacaaatcaggaaaccgag ggtcaagattgttgctcctc cgctctcatcatactcttgc agcaatatcgtactcctgaag atgctgagaaagtctttgctg doi:10.1371/journal.pone.0030472.t003 The asf1-33 mutant required Rad3 and Chk1 kinases for cell cycle arrest and survival The cell elongation Solithromycin phenotype and phosphorylation of Cdc2 led us to test checkpoint activation in the asf1-33 mutant. As cell cycle checkpoint pathways frequently consist of a set of protein kinases, we considered the possible involvement of novel protein kinases. To that end, we used a deletion set of protein kinases constructed by M. Balasubramanian to generate double mutants with asf1-33 mutant by mating. Some deletion strains that were deficient in MAP kinases were excluded from this experiment because MAP kinases are unlikely to be involved in the mitotic cell cycle checkpoint. Strains lacking 77 different kinases were mated with the asf1-33 mutant on nitrogen-free EMM2 to construct double mutants. Random spore analysis was performed, and double mutants lacking a specific kinase gene and possessing the asf1-33 mutation were selected by G418 resistance and uracil auxotrophy. Cell morphology and viability of the 77 strains was examined after incubation at 36uC for 24 h on YES medium. The phenotypes of each double mutant were categorized into four types: not elongated and enhanced lethality, no