Extended Data Fig. 4: Characterization of Slx4∆SAP/– and Slx4∆SBD/– mutant clones.
a. Strategy for in-frame deletion of SAP domain-encoding region of Slx4. Red half-arrow heads: genotyping PCR primers. Gels show PCR products using gDNA from Slx4+/– and Slx4∆SAP/– clones. Sequencing chromatogram shows in-frame breakpoint of Slx4∆SAP allele. b. RT-qPCR analysis of UBZ, MLR, SAP and SBD domains encoding mRNA in Slx4+/– and Slx4∆SAP/–clones. Data normalized to Gapdh mRNA using the 2–ΔCT method, mRNA expression in Slx4∆SAP/– were normalized to Slx4+/–. 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. Error bars: s.d. P = 0.0478 for mRNA expression of the SAP region in Slx4+/– compared to Slx4∆SAP/–. No significant differences were observed in expression levels of all other domains. c. Anti-HA immunoblot of two independent HA-degron tagged Slx4∆SAP/– and Slx4+/– isogenic cell lines. The parental Slx4+/– untagged cell line was used as a control. d and e. Tus/Ter-induced LTGC (d) and I-SceI-induced LTGC (e) in Slx4+/– and Slx4∆SAP/– clones. Data shows mean values of five biologically independent replicates, n = 5. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. Error bars: s.e.m. f. Strategy for in-frame deletion of SBD domain-encoding region of Slx4. Red half-arrow heads: genotyping PCR primers. Gels show PCR products using gDNA from Slx4+/– and Slx4∆SBD/– clones. Sequencing chromatogram shows in-frame breakpoint of Slx4∆SBD allele. g. RT-qPCR analysis of UBZ, MLR, SAP and SBD domains encoding mRNA in Slx4+/– and Slx4∆SBD/–clones. Data normalized to Gapdh mRNA using the 2–ΔCT method, mRNA expression in Slx4∆SBD/– was 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. P = 0.048 for mRNA expression of the SAP region in Slx4+/– compared to Slx4∆SBD/–. No significant differences were observed in expression levels of all other domains. h. Anti-HA immunoblot of two independent HA-degron tagged Slx4∆SBD/– and Slx4+/– isogenic cell lines. The parental Slx4+/– untagged cell line was used as a control. i and j. Tus/Ter-induced LTGC (i) and I-SceI-induced LTGC (j) in Slx4+/– and Slx4∆SBD/– clones. Data shows mean values of five biologically independent replicates, n = 5. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. Error bars: s.e.m.
