Fig. 3

Chromatin compaction threshold restricts excessive loading of replication licensing factors. a U2OS cells were synchronized with double thymidine block, transfected with either siControl or siSET8 6 h before G1/S release, and fixed at 15 h post release. Cells were pre-extracted in CSK buffer containing 0.5% triton and immunostained with the indicated antibodies. Cells were counterstained with DAPI (Bar, 10 µm). b Scatter plot showing the quantification of ORC1 intensity from cells in a where mean ± SD is indicated by red lines. n > 150, ****p < 0.0001 (unpaired t test). n > 280. c Scatter plot showing the quantification of MCM2 intensity from cells in a where mean ± SD is indicated by red lines. n > 150, ****p < 0.0001 (unpaired t test). n > 280. d Chromatin fraction was prepared from synchronized U2OS cells depleted as in a, harvested at 15 h, and immunoblotted with the indicated antibodies. e Chromatin was prepared from cells synchronized and transfected with siRNAs as in a, treated with sucrose at 12 h post G1/S release, and harvested at 15 h. Samples were blotted with the indicated antibodies. f U2OS cells expressing DOX-inducible FLAG-HA-tagged histone H4WT or H4K20A mutant were synchronized as in a and fixed at 15 h post release. Cells were pre-extracted and immunostained with the indicated antibody as well as DAPI for DNA (Bar, 10 µm). g Scatter plot showing the quantification of ORC1 intensity from HA-positive cells in f where mean ± SD is indicated by red lines. n > 250, **p < 0.01 (unpaired t test). h U2OS cells expressing DOX-inducible FLAG-HA-tagged histone H4WT or H4K20A mutant were synchronized as in a and fixed at 15 h post release. Cells were pre-extracted and immunostained with the indicated antibody as well as DAPI for DNA (Bar, 10 µm). i Scatter plot showing the quantification of MCM2 intensity from HA-positive cells in h where mean ± SD is indicated by red lines. n > 180, ****p < 0.0001 (unpaired t test)