Fig 2: Cell proliferation and DNA replication changes in the ORC1-/- cell lines.(A) Growth curves of indicated clones of cells over four days, expressed as MTT absorbance relative to the level at day 1. (Mean ± S.D.; n = 4 biological replicates) Cells after passage for 1 month or six months. (B) FACS profile of propidium-iodide stained cell-cycle asynchronous cells from indicated clones. (C) Cells arrested in double-thymidine block released into nocodazole containing medium and harvested at indicated times after release to measure rate of progression through S phase. AS: asynchronous cells. The red dotted lines indicate cells with G1 and G2 DNA content. (D) Molecular combing of chromosomal DNA after a pulse of CldU for 30 min chased with a pulse of IdU for 30 min. Box and whiskers plot for fork progression rate and inter-origin distance of indicated clones of cells. (P value < 4.6e-06, two-sided Wilcoxon rank sum test for two samples N = number of tracks counted) (Inter origin disntance N = 91(WT), 131(ORC1B14), 174(ORC1BP32) p: Statistical significance of any difference between WT and ORC1-/- cells. (E) Overlap of the BrIP-seq peaks between WT and ORC1-/- cells. (F) Box and whiskers plot for inter-origin distances (measured by BrIP-seq) for each chromosome in WT and ORC1-/- cells. The median inter-origin distance for all chromosomes together indicated at bottom right. (G) Circos plot of Origins mapped by BrIP-seq for chromosome 1. Outer circle: the chromosome with the karytotyping bands. Inner two circles: the locations of BrIPseq peaks in the WT and ORC1-/- cell lines. (H) Distribution of BrIP-seq mapped origins in ORC1-/- cells relative to distance from Transcription Start Sites (TSS).DOI: http://dx.doi.org/10.7554/eLife.19084.006