Extended Data Fig. 6: Characterization of Slx4∆MLR/– mutant clones. | Nature Structural & Molecular Biology

Extended Data Fig. 6: Characterization of Slx4∆MLR/– mutant clones.

From: The structure-specific endonuclease complex SLX4–XPF regulates Tus–Ter-induced homologous recombination

Extended Data Fig. 6

a. Strategy for in-frame deletion of MLR domain-encoding region of Slx4. Red half-arrow heads: genotyping PCR primers. Gels show PCR products using gDNA from Slx4+/– and Slx4∆MLR/– clones. Sequencing chromatogram shows in-frame breakpoint of Slx4∆MLR allele. b. RT-qPCR analysis of UBZ, MLR, SAP and SBD domains encoding mRNA in Slx4+/– and Slx4∆MLR/–clones. Data normalized to Gapdh mRNA using the 2–ΔCT method, mRNA expression in Slx4∆MLR/– were normalized to Slx4+/– of the same experiment. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n = 3). Analysis by unpaired Student’s t-test (n = 3). Error bars: standard deviation (s.d.). P = 0.01 for mRNA expression of the MLR region in Slx4+/– compared to Slx4∆MLR/–. No significant differences were observed in expression levels of all other domains. c Anti-HA immunoblot of two independent HA-degron tagged Slx4∆MLR/– and Slx4+/– isogenic cell lines. The parental Slx4+/– untagged cell line was used as a control. d-g. Quantification of cell survival assays of Slx4+/– and Slx4∆MLR/– clones in the presence of MMC (d), Olaparib (e) 5-aza-2′-deoxycytidine (f) Zebularine (g), Hydroxyurea (h) and Camptothecin (i). Data show mean values of three biologically independent replicates, n = 3. P-value: *p < 0.05. Analysis using unpaired Student’s t-test. Error bars: s.d. j. Representative images of metaphase spreads showing chromatid breaks (black arrows) and radial chromosomes (black arrowheads) in cells either untreated or treated with 20 ng/mL MMC for 12 h in Slx4∆SAP/– and Slx4∆SBD/– cells. k. and l. Quantitation of the number of breaks (k) and radial chromosomes (l) per metaphase nucleus in Slx4 mutant cells shown. Treated and untreated cells were harvested at the same time and each sample represented one independent experiment, n = 1. Data shows mean values of breaks or radials per genotype. Analysis using unpaired Student’s t-test. P-value: ****p < 0.0001.

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