These results also suggest that there may be a synergistic effect of Hes 1 SUMO

Cytotoxicity increased steeply with the length of contact and reached completion after 6 min only. More prolonged incubation did not result in increased cell death. Therefore, ARQ 197 c-Met 阻害剤 the length of contact with NCS was 10 min throughout. For studies of H2O2 response, serial dilutions of concen trated H2O2 were made in pure water, then in cul ture medium immediately prior to use. The length of contact with H2O2 was 10 min. In some experiments ANI was used as a PARP 1 inhibitor. When present, ANI from a 3 mM stock solution in pure DMSO was introduced 1 h prior to irra diation or NCS, and removed 1 h later with two HBSS washes. The DMSO concentration in medium was 1% and was kept constant throughout experiments with ANI. Controls were made with DMSO alone at the same concentration.<br><br> DNA double strand break determination The determination of DSB formation and repair was car ried out by pulsed field agarose gel electrophoresis through the clamped homogeneous electric field technique. Thymidine AZD0530 Sr 阻害剤 labeled cells were put in ice following treat ment, harvested in ice cold PBS supplemented with 2 mM EDTA by gentle scraping, and collected by sedimentation at 4 C. Pellets from 5 105 cells were resuspended in 150 l, final volume, of PBS buffer at 37 C, and mixed under mild vortexing with an equal volume of 1. 6% w v low melting point agarose in PBS buffer at 37 C. The suspension was immediately pipetted into pre chilled 4 10 mm moulds and allowed to form plugs on ice for 30 min. Embedded cells were subse quently lysed by immersion of the plugs in 2 ml of a solu tion containing 2% N lauroyl sarkosine, 40 mM EDTA, 1 mg ml proteinase K in PBS, pH 7.<br><br> 8 and incubated, firstly for 2 h in ice, secondly for 24 h at 50 C. The plugs were subsequently washed twice with PBS, and treated for 1 h with 200 g ml RNase A. The plugs were finally washed twice with PBS and stored at 4 C overnight prior electrophoresis. The plugs were inserted into the wells of an 0. 8% w v aga rose gel made in 0. 75 TAE buffer, and the gels submitted to PFGE at 14 purchase Alvocidib C in a CHEF DR III apparatus with buffer recirculation at 14 C. Migration was for 72 h at 2 V cm with three switch times, namely, 1200 s, 1500 s and 1800 s with angles of 96, 100 and 106, respectively. The molecular weight mark ers were S. pombe and S. cerevisiae chromosomes. After electrophoresis, the gels were stained with 0.<br><br> 5 g ml ethidium bromide under mild agitation, fol lowed by destaining for 1 h in fresh buffer. DNA fluores cence was visualized over an UV transilluminator with camera recording. The gels were subsequently dried in vacuo over a piece of Whatman paper and analyzed using a Phosphorimager apparatus, allowing a precise determination of the migration profile of DNA fragments and of the fractional radioactiv ity released from the plugs. Immunofluorescence of PARP 1, pADPr and H2AX 3T3 fibroblasts were grown for 24 h on coverslips and exposed to either rays, H2O2, or graded concentrations of NCS, and fixed 10 min after the beginning of treatment. For PARP 1 and pADPr immunofluorescence, fixation was by 4% formaldehyde in PBS followed by two PBS washes and neutraliza tion of residual formaldehyde by 50 mM NH4Cl. After a further PBS wash, cells were permeabilized by 0.