Fig. 2: Resection oscillates following a circadian pattern.

A CRY1 (siCRY1) or control (siNT) depleted U2OS cells were transfected with the human version of CRY1 (black bars) or an empty vector (white). 1 h after irradiation with 10 Gy cells were immunostained for RPA. Then, the percentage of cells with more than 10 RPA foci was scored (left). The average and standard deviation of three independent experiments is shown. Statistically significance was calculated using a two-way ANOVA. Only statistical different comparisons are depicted. Representative images are shown on the right side. Scale bar 25 µm. B Same as Fig. 1D, but in cells transfected with an siRNA against CRY1 or a control sequence, as indicated. Cells were irradiated every 4 hours after release in order to distinguish between a delay or a general upregulation of resection. C Average recruitment of RPA at the 214 best AsiSI cutting sites measured by ChIP-seq in samples transfected with an siRNA against CRY1 or a control sequence, as indicated (solid lines). One representative experiment, out of 2 with similar results, is shown. Similar analysis was performed in 200 random sites (dashed lines). D QIBC representation of RPA foci formation. Cells treated as in A were immunostained for RPA. The intensity of RPA or DAPI were plotted in the Y and X axis, respectively. The vertical dashed line represents the threshold of DAPI intensity corresponding to S/G2 cells, whereas the horizontal line represents the threshold for RPA positive cells. The percentage of S/G2 cells showing RPA foci signal is indicated. A representative experiment out of 2 two independent replicates is shown. E Same as A but in cells overexpressing or not CRY1 without siRNA transfection. The average and standard deviation of three independent experiments is shown. F Same as A but in cells treated with the CRY1 stabilizer KL001. The average and standard deviation of three independent experiments is shown. G Gene conversion was assessed in U2OS cells bearing the DR-GFP reporter in cells transfected with an siRNA against CRY1 (siCRY1) or a control sequence (siNT), as indicated. The amount of GFP-positive cells, i.e., cells that have undergone repair by gene conversion, after induction with the nuclease I-SceI, was normalized to the siNT control taken as 1. The statistical significance was calculated using a Student´s t-test. The average and standard deviation of four independent experiments is shown. H Same as G, but using the SSA reporter SA-GFP. I Same as G, but using the NHEJ reporter EJ5-GFP. J Same as G, but in cells bearing an empty vector or a vector harboring an ectopic version of CRY1. The average and standard deviation of four independent experiments is shown. K Same as J, but using the SSA reporter SA-GFP. L Same as J but using the NHEJ reporter EJ5-GFP. M RAD51 foci 3 h after exposure to irradiation were scored by immunofluorescence in cells depleted for CRY1 (siCRY1) or control cells (siNT). The number of RAD51 foci per cell was counted and represented as a single dot. The median number of Rad51 foci is shown as a red line. Statistical significance was calculated using a Mann–Whitney test. A representative experiment out of three with similar results is shown. N Same as M, but in cells transfected with a plasmid bearing an ectopic copy of CRY1 or an empty vector. O Same as M, but immunostaining for RIF1. P Same as N, but immunostaining for RIF1. Q U2OS cells transfected with an siRNA against CRY1 (siCRY1), CCAR2 (siCCAR2), both (siCRY1siCCAR2) or a control sequence (siNT), were irradiated with 10 Gy. Then, the percentage of cells with more than 10 RIF1 foci was scored at the indicated time points after irradiation. The average and standard deviation of three independent experiments is shown. R Same as M, but immunostaining for 53BP1. S Protein samples, depleted or not for CRY1 as indicated, were taken 1 h after irradiation. Upon cellular fractionation, both the whole cell extract (WCE) and the chromatin-bound fraction (Chromatin) were resolved in SDS-PAGE and blotted with antibodies against the indicated proteins. A representative experiment out of three replicas with similar results is shown. For the whole figure, source data are provided as a Source Data file.