Artifacts arising from fork-to-fork fusion, cells had been pulsed with BrdU for ten min within the absence of HU and 20 min within the presence of HU to attain similar replication fork length. (A) Examples of a DNA fiber containing a replicon cluster of 4 BrdU-labeled forks are shown. (B) Distribution on the mean intra-cluster fork spacing from 50 replicon clusters is shown. General fork spacing SEM is indicated within the chart. (C ) Comparisons among CCE cells derived from the 129/Sv mice and NSPCs from the E13.5 129/Sv embryo brains. (C) Immunoblotting of chromatin-bound MCM proteins with H3 as a loading handle for quantification is shown. (D) Quantification of chromatin-bound MCM2 in G1phase cells and cell-cycle distribution by FACS are shown. (E) 2D projection confocal and SIM images of chromatin-bound MCM2, MCM3, and MCM7 in G1 phase cells are shown. (F) Quantification of chromatin-bound MCM foci number and average focus volume imaged by SIM are shown. Error bars represent SEM of three independent experiments. (G) DNA fiber analysis of NSPCs and ESCs is shown. Cells have been incubated with 100 mM HU for four hr just before BrdU pulse. General fork spacing SEM from 50 replicon clusters is indicated. p values are from two-tailed t test.1k 800 Histogram 600 400 200 0 01k 800 600 400 200frequency0.3 frequency0.0.0 0 two four 6 0 ten 20 30 40 50 imply intra-cluster fork spacing (kb)DNA content (arbitrary units)Econfocal3D-SIMF3000 foci number by SIMESC NSPCESC2500 2000 1500 1000 500 0 MCM2 MCM3 MCM2average foci volume3000 2500 2000 1500 1000 500 0 MCM2 MCM3 MCMNSPC2Stem Cell Reports j Vol. 5 j 18594 j August 11, 2015 j 015 The AuthorsABDCEFH GILJ K(legend on subsequent page)188 Stem Cell Reports j Vol. five j 18594 j August 11, 2015 j 015 The Authorscontrols (Figures 2L and S2E). Together, these data suggest that, upon reduction of DOs, ESCs sustain normal selfrenewal but are impaired in differentiation. This is constant with our observation that ESCs load a lot more DOs than NSPCs. Consequently, the self-renewal of ESCs is much more robust against DO reduction than differentiation. Lowering DOs Impairs ESC Reversible Inhibitors products differentiation to NSPCs We additional investigated the differentiation of the Mcm4C/C ESCs into NSPCs. Mcm4C/C ESC-derived NSPCs show hyper-activation of phosphorylated CHK1, P53, and H2AX and elevated apoptosis (CASPASE three cleavage and 3-fold increase in TUNEL staining; Figures 3A, 3B, and S3A 3C). Addition of caffeine, an ATM/ATR inhibitor, or the CASPASE inhibitor Z-VAD-FMK through NSPC differentiation largely rescued the differentiation efficiency, as shown by the increased expression of NESTIN and SOX1 (Figures 3C and S3C). The partial nature from the rescue could possibly be as a result of essential role of ATR kinase throughout DNA replication and cell-cycle progression (Jirmanova et al., 2005; Ruzankina et al., 2007). Despite this, the above information clearly illustrate a functional relationship among lowered DOs and impaired neural differentiation from the Mcm4C/C ESCs due to elevated DNA harm response and cell death. The defect within the neural differentiation with the Mcm4C/C ESCs is probably resulting from compromised survival of differentiating cells. To confirm our in vitro findings on neural differentiation, we isolated NSPCs in the Mcm4C/C mice in the course of embryogenesis. NSPCs from the forebrain on the E13.5 Mcm4C/C embryos generated 50 fewer neurospheres than the wild-type NSPCs, although both expressed equivalent amount of NESTIN and SOX2 (Figures 3D, S3D, and S3E). Moreover, NSPCs in the Mcm4C/C embryos.