Fig. 2

Role of meiotic cohesins in replication fork progression. a,d DNA fiber labeling scheme (Top): IdU is incorporated at the first analog (15 min), followed by CldU (15 min), which was incorporated as the second analog. Representative image of DNA fiber in ESCs and MEFs (bottom). Scale bars are 2.5 μm. b,e Quantification of length of IdU and CIdU tracks in siCtrl and siCohesin. The cells were transfected with siCtrl or siCohesin (siSMC3, siRAD21, siREC8, and siSTAG3) before DNA labeling, as indicated in the figure. Each biological replicate is color-coded (red, green, and blue) and the average of each replicated data is indicated with a larger dot, and black bars indicate the averages of three means (n ≥ 40 for condition). The error bars are the mean ± SD from three independent biological replicates. Statistics: Paired two-tailed t-test; ns, not significant; ***P < 0.001. c,f Quantification of elongation rates during DNA replication. The lengths of the DNA fibers (IdU and CldU tracks) were measured as shown in a. To calculate the DNA fork rates, the DNA fiber lengths were converted to kilobases (kb) (1 mm = 2.94 kb) and the converted values were divided by the CldU/IdU pulse labeling time (30 min). Statistics: Paired two-tailed t-test; ns, not significant; ***P < 0.001; n, the number of measured fibers. g,h Images of RAD51 and RPA foci formation in ESCs and MEFs (top). The focus size in each condition was analyzed using Nikon software (bottom). Scale bars = 2.5 μm. i,j Quantification of RAD51 and RPA foci. The numbers of focus were counted in siCtrl (control) cells and siCohesin (siSMC3, siRAD21, siREC8, and siSTAG3)-treated cells. The error bars are the mean ± SD from three independent experiments (n ≥ 50 for condition). k,l Average focus size after knockdown of cohesin components. Focus size was quantified in normal cells and cohesin knockdown cells. The error bars are the mean ± SD from three independent experiments (n ≥ 50 for condition)