Fig. 2.

OBI1 is required for replication origin firing. a Involvement of OBI1 in cell proliferation. U2OS cells were transfected with siRNA pools targeting OBI1 3′UTR (siUTR) or coding sequence (siOBI1), ORC1 (siORC1), CDC7 (siCDC7) or a non-targeting siRNA (siMock) (sequences in Supplementary Table 3). Cell proliferation (fold-increase relative to day 0) was evaluated by counting cells every day after transfection. The mean results of three independent experiments are shown. Expression of endogenous OBI1, ORC1, CDC7 and PCNA was monitored by western blotting at day 3 (right). b U2OS cells were transfected with siRNAs as in a. Three days post-transfection, cells were incubated with BrdU for 15 min. BrdU incorporation and DNA content were analysed by flow cytometry (left panels). Lines delimiting BrdU-positive siMock-treated cells are shown. BrdU incorporation fluorescence signal was quantified from three independent experiments (right panel). c U2OS cells were transfected with siRNAs as in a. Three days post-transfection, cells were incubated with IdU (20 min) followed by CldU (20 min) and processed for DNA combing analysis (see Methods). Representative images of bidirectional forks labelling are shown. d Analysis of replication fork speed (in kb/min) in the cells described in c, based on the measurement of CldU tracks preceded by the IdU signal (two independent experiments). Red bars indicate median values. e Inter-origin distances (in kb) in the cells described in c were quantified from two independent experiments. Red bars indicate median values. f The mean global fork density (in fork/Mb) in the cells described in c was quantified by measuring the number of labelled forks per megabase of combed DNA, normalised to the percentage of S-phase cells (two independent experiments). g U2OS cells were transfected with siRNAs as in a. Three days later, chromatin and soluble fractions were isolated and analysed by western blotting with antibodies against the indicated proteins. *p < 0.01; **p < 0.001