Involvement of the cohesin complex in HR-mediated DNA repair mechanism. (A-D) α-Kleisin factor and RPA staining in ESCs and MEFs. ESCs and MEFs were immunostained against RPA and REC8 (A, C) and against RPA and RAD21 (B, D). ESCs and MEFs were untreated (normal condition) (A, B) and REC8 or RAD21 were depleted in ESCs (C, D). (E, F) RPA focal formation in REC8- or RAD21-depleted MEFs. (G, H) Quantification of cohesin intensity. ESCs were treated with siRNA against REC8 or RAD21, after which the intensity of each kleisin factor was measured. The error bars indicate mean ± standard deviations (SD; n = 40 for each experiment). The number of RPA foci was quantified in kleisin-depleted ESCs (I) or MEFs (J) treated with siREC8 or siRAD21 (n = 120, three independent experiments for ESCs; n = 80, two independent experiments for MEF). (K) Schematic of the treatment of cells with siRNA against REC8 or RAD21 and hydroxyurea (HU). (L, M) Increase in the intensity of REC8 or RAD21 and RPA focal formation in DNA-damaged and DNA-depleted ESCs. (N) Measurement of RPA focal number. The blue, green, and orange bars indicate the average of each experiment (n = 3). The values represent the RPA foci per nucleus of 40 cells per experiment.
