Extended Data Fig. 2: The SLX4 UBZ domain promotes resistance to DPC-inducing drugs.
a. Strategy for in-frame deletion of UBZ domain-encoding regions of Slx4. Red half-arrow heads: genotyping PCR primers. Gel shows PCR products using gDNA from Slx4+/– and Slx4∆UBZ/– clones. Sequencing chromatogram shows in-frame breakpoint of Slx4∆UBZ allele. b. RT-qPCR analysis of mRNA encoding UBZ, MLR, SAP and SBD domains in Slx4+/– and Slx4∆UBZ/–clones. Data normalized to Gapdh mRNA using the 2–ΔCT method, mRNA expression in Slx4∆UBZ/– samples 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. P = 0.0125 for mRNA expression of the UBZ region in Slx4+/– compared to Slx4∆UBZ/–. No significant differences were observed in expression levels of all other domains. c-h. Quantification of colony formation of Slx4+/–, Slx4∆UBZ/– and Brca1-∆exon11 clones in the presence of MMC (c), Olaparib (d), 5-aza-2′-deoxycytidine (e) Zebularine (f), Hydroxyurea (g) and Camptothecin (h). Data shows mean of values of three biologically independent replicates, n = 3. Analysis using Student’s t-test. P-value *p < 0.05 and ***p < 0.001. Red asterisks refer to comparison between Slx4+/– and Slx4∆UBZ/– clones; blue asterisks denote comparison between Slx4+/– and Brca1-∆exon11 clones and blue bracket with red asterisks denotes comparison between Slx4∆UBZ/– and Brca1-∆exon11 clones. Error bars: s.d. i. RT-qPCR analysis of N-terminal Flag-tagged wild-type full-length human SLX4 and UBZ 4 C > A expression plasmids 48 h after transfection. Data normalized to Gapdh mRNA using the 2–ΔCT method and compared to empty vector (EV) control. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n = 3). Error bar: s.d. j. Western blot analysis of the chromatin-bound fraction of various human 3xFLAG-SLX4 full-length and UBZ 4 C > A transiently expressed for 48 hours and blotted with anti-FLAG antibody. k. Western blot analysis of whole cell extract (WCE) and chromatin-bound fraction of clones of Slx4+/– and Slx4∆UBZ/– tagged with a dual degron containing a C-terminal 8xHA tag and untagged hemizygote cells (+/–). Asterisk (*) denotes non-specific bands.
