First transfection, cells were treated with 134 nM gemcitabine for 65 h. For methylation-sensitive PCR of HpaII site pOctTKEGFP 2299 cells in Figure 6 well plates were transfected 3. DNA demethylation mediated Gadd45a is not affected by inhibitors of BER. The methylation status of HpaII site in the regulatory region of 2299 GDC-0980 PI3K inhibitor pOctTK EGFP was analyzed by methylation-sensitive PCR 48 h after transient transfection with or without co hGadd45a. The cells were treated with the topoisomerase II inhibitor etoposide, NER inhibitors gemcitabine and camptothecin or BER inhibitors CRT 0044876, betulin Acid and ABT 888 as shown. Untransfected methylated and unmethylated HpaII was in vitro reporter plasmid used as reference. P, 0.05, p, 0.01, p, 0.
001: The significance was determined by unpaired Student, St-test using the untreated sample transfected Gadd45a judged as a reference. doi: 10.1371/journal.pone.0014060.g003 Figure 4 Gemcitabine inhibits DNA synthesis Au Erplanm Strength DNA methylated. Oct4 GSK256066 801312-28-7 plasmid was methylated with or without BrdU in Xenopus oocytes injected in the presence or absence of gemcitabine and recovered after the incubation. Controlled for L equal loading in vitro BrdU-labeled luciferase plasmid was added after lysis of the oocytes. PCR analysis of immunpr Zipitierten DNA with specific primers oct4 or Luc performed. doi: 10.1371/journal.pone.0014060.g004 GEMZAR demethylation Bl CKE PLoS ONE | www.plosone fifth November 2010 | Volume 5 | Issue 11 | e14060 with 100 ng of the contr PBL hGadd45a KS plasmid or with 200 ng of EGFP pOctTK Turbofect transfection reagent according to manufacturer’s instructions.
Immediately after transfection, cells were incubated with 50, 100 or 150 nM gemcitabine, 15, 25 or 50 nM camptothecin, 50, 100 or 200 mm CRT 0044876, 1, 5 or 10 mM betulin Acid, 5, 10 treated 20 mm or 888 or ABT 10, 20 or 40 nM for 48 h etoposide. Luciferase reporter assay of duplicate tests were performed luciferase reporter 40 h after transient transfection of the HEK293T cell DNA in 96-well plates, with a total amount of 110 ng of DNA per well, containing 5 ng luciferase reporter firefly, PBS or 5 ng 5 ng of Xenopus tropicalis Gadd45a plasmid, 0.1 ng Renilla luciferase reporter plasmid and 100 ng of PBS. Reporter plasmids were produced in bacterial strain SCS110 dam2/dcm2 and methylated in vitro with HpaII and HhaImethylase.
The transfections were performed in triplicate. Optionally, the cells were treated with 67 nM gemcitabine, 26 nM camptothecin, etoposide 43 nM, 30 nM or 20 nM b lapachone merbarone 18 h The results are presented as the mean of triplicate and error bars indicate the standard deviation. The experiments were repeated three times. A quantitative RT-PCR-RNA was reverse transcribed using the RNeasy kit and with Superscript II reverse transcriptase. Real-time PCR was performed using Roche LightCycler480 mean Be probes and primer-probe in combination with mono-hydrolysis of predefined color Roche universal probe library. The following primers and probes were con UPL Us on applied science.com / SIS / rtPCR / UPL / adc.jsp hMLH1 GAATGCGCTA TGTTCTATTCCA before 59, 59 Conversely 38th ATGGAGCCAG GCACTTCA, UPL probe No.
Was used for quantification Roche LC480 module relative quantification software. All values were normalized to the level of the housekeeping gene GAPDH. Analysis of the genomic DNA methylation of DNA from cells treated or reporter plasmids were transfected using the kits in blood and tissues. The DNA was divided into three parts and is digested with PvuII, HpaII or its methylation-insensitive isoschizomer MspI. The methylation was compared by comparing HpaII digested samples determined contr The DNA digested with PvuII qPCR with methylation-sensitive PCR primers. Was performed as an internal control normalization, a PCR with primers for methylation-sensitive. MspI digest was used as a control for the restriction enzyme intact Figure 5 Gemcitabine has no influence on the global methylation levels. The methylation of chromosome 1 satellite 2 in HEK293 cells or MCF7 was