Fig. 5: CRY1 levels modulate the response to DNA damaging agents in vitro.

A U2OS transfected with an siRNA against CRY1 or a control sequence were seeded at low density and irradiated at the indicated dose. Cells were left to grow for 10 days and the survival fraction, compared with an unirradiated sample taken at 100%, is plotted. The average and standard deviation of three independent experiments is shown. Statistically significance was determined using a one-way ANOVA. B Same as A but in cells treated with the indicated doses of etoposide (VP16) for 1 hour. The average and standard deviation of three independent experiments is shown. Statistically significance was determined using a one-way ANOVA. C Same as A but in cells pre-exposed to the CRY1 stabilizer KL001 or DMSO. The average and standard deviation of three independent experiments is shown. Statistically significance was determined using a one-way ANOVA. D Same as B but in cells pre-exposed to the CRY1 stabilizer KL001 or DMSO. The average and standard deviation of three independent experiments is shown. Statistically significance was determined using a one-way ANOVA. E Same as A, but in cells synchronized with DEX, released for the indicated time and exposed to 2 Gy of IR. The average and standard deviation of three independent experiments is shown. Statistically significance was determined using a one-way ANOVA. F U2OS cells depleted or not of CRY1, as indicated, were irradiated with 10 Gy. 24 h after irradiation cells were stained with DAPI and the percentage of cells with micronuclei was calculated. The average and standard deviation of two independent experiments is shown. Statistically significance was determined using a one-way ANOVA. G Percentage of chromosome breaks present in mitotic cells upon irradiation with 2 Gy transfected with the indicated siRNAs (left) or exposed to KL001 or DMSO (right). H Number of γH2AX foci in U2OS cells unchallenged (−) or 24 h after irradiation (+; 10 Gy), in cells transfected with an siRNA against CRY1 (+) or a control sequence (−), as indicated. A representative experiment out of three is shown. Statistical significance was analyzed using an unpaired one-sided Student’s T-test I Same as A but in cells depleted for CRY1, CCAR2 or both proteins simultaneously. The average and standard deviation of three independent experiments is shown. J Same as B but in cells depleted for CRY1, CCAR2 or both proteins simultaneously. The average and standard deviation of three independent experiments is shown. K Same as F but in cells depleted for CRY1, CCAR2 or both proteins simultaneously. The average and standard deviation of three independent experiments is shown. Statistically significance was determined using a one-way ANOVA. L Cells treated as in panel K were assayed for DNA break presence using neutral comet assay. Left, quantification of one out of three similar experiment. Each dot represents one nucleus. Right, a representative image of a nucleus for each condition is shown. A representative experiment out of three is shown. Statistical significance was analyzed using an unpaired one-sided Student’s T-test. For the whole figure, source data are provided as a Source Data file.