Specifically, LY294002 administration resulted in a dose dependent decrease

Data was acquired on an LSR II flow cytometer 17-AAG CP 127374 using the FACS DIVA software. All flow cytometry data were analyzed with FlowJo or WinList. Daily QC of the LSRII cytometers was performed as previously described. Dead cells and debris were excluded by forward and side scatter proper ties combined with amine aqua viability dye exclusion. For AML samples, all non apoptotic leukemic cells were identified based on expression of CD45 and side scatter properties and lack of the apoptosis marker cleaved PARP as previously described, while CyclinA2 staining discriminated CyclinA2 and CyclinA2 subsets. Similarly, normal lym phocytes within AML samples were identified by low side scatter and high CD45 expression as previously described.<br><br> For cell lines, forward scatter, side 17-DMAG HSP-90 阻害剤 scatter, amine aqua, and cleaved PARP similarly identified live non apoptotic cells and CyclinA2 staining discrimi nated CyclinA2 and CyclinA2 subsets. Specific drug treatments and readouts examined were as followsa For experiments measuring multiple DDR readouts after etoposide treatment, cell lines or primary AML samples were treated with 30 ugmL eto poside for 2 h or 6 h and assayed for p BRCA1. pDNA PKcs. p 53BP1, p ATM, p p53, p Chk2, and p H2AX. b For experiments showing magnitude and reproduci bility of multiple AZD2281temozolomide induced DDR readouts and the ability of these readouts to stratify for HRR status, BRCA1, BRCA1. and BRCA2 cell lines were treated with 6 ugmL AZD2281 2 ugmL temozolo mide for 48 72 h and assayed for p H2AX, p RPA232. p DNA PKcs, p21, p ATM and p BRCA1 in CyclinA2 and CylcinA2 cells.<br><br> In these experiments a 2 ugmL dose of temozolomide was used due to excessive apoptosis observed with the combination of AZD2281 with higher doses of temozolomide at later timepoints. A66 1166227-08-2 c For experiments measuring the cell cycle selectivity of individual genotoxic agents, cell lines in culture or AML samples pre treated for 48 h with a panel of myeloid growth factors to induce proliferation in all AML subtypes were challenged for 6 h with etoposide, PARP inhibitor AZD2281, temo zolomide, or the combination of AZD2281 temozolomide and assessed for in duced p H2AX in distinct subsets of cells including CyclinA2 cells, CyclinA2 cells, or All live, cPARP cells regardless of CyclinA2 status.<br><br> Data analysis details Metrics Metrics, illustrated in Figure 2, for quantifying DDR were applied to gated cell populations con taining at least 200 events and have been described previously. Briefly, the Log2Fold metric measures the magnitude of the responsiveness of a cell population to modulation where a value of zero indicates lack of induced signaling while a positive value indicates an in crease in the signaling response of a population. The Uu metric measures the proportion of responsive cells by comparing the overlap and rank order of the modulated and unmodulated populations on a cell by cell basis and ranges from zero to one where 0. 5 indicates no change, values 0. 5 indicate cells have increased in signal, and values 0. 5 indicate that cells have decreased in signal vs. an unmodulated population.