Extended Data Fig. 8: Characterization of Xpf+/– and Xpf∆Nuc/– cells.
a. CRISPR/Cas9 with dual sgRNA strategy for generation of Xpf+/– hemizygous cells. The Xpf– allele contains a 42.3 kb deletion between the 5’UTR and exon 11. Red half arrowheads: PCR and sequencing primers specific to 5’UTR and Exon 11. Predicted PCR product sizes for Xpf+ (wild type) allele shown. b. Gel shows PCR products using gDNA from Xpf+/+ and Xpf+/– clones. Note the 440 bp PCR product formed across the 42.3 kb deletion between the 5’UTR and exon 11 in Xpf+/– cells. c. RT qPCR analysis of mRNA in Xpf+/+ and Xpf+/– cells. Data shows mean of three independent experiments (n = 3) normalized to Gapdh mRNA using the 2–Δ∆CT method and analyzed by unpaired Student’s t-test. Error bars: s.d. d. Western blot of whole cell lysates in Xpf+/+ or Xpf+/– clones. siLuc and siErcc1 were compared in Xpf+/– cell line 48 hours after transfection to validate specificity of the Ercc1 band. e. and f. Quantification of colony formation of Xpf+/+ vs. Xpf+/– clones in the presence of MMC (e), Olaparib (f). Data show mean values of three biologically independent replicates, n = 3. Error bars: s.d. g. Tus/Ter-induced and I-SceI-induced HR frequencies in Xpf+/+ or Xpf+/– clones. Data shows mean values of four biologically independent replicates, n = 4. Error bars: s.e.m. Analysis by unpaired Student’s t-test. Error bars: s.e.m h. RT-qPCR analysis of mRNA encoding the XPF helicase, nuclease and Hh2h domains in 4 isogenic Xpf +/– clones and 4 Xpf∆Nuc/– clones. Data normalized to Gapdh mRNA using the 2–ΔCT method, mRNA expression in Xpf∆Nuc/– was normalized to Xpf +/– 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. Error bars: s.d. P = 0.0153 for mRNA expression of the Nuclease region in Xpf+/− compared to Xpf∆Nuc/–. No significant differences were observed in expression levels of all other domains. i. Western blot analysis of ERCC1 in whole cell extract (WCE). *: non-specific band. β-tubulin: Loading control. j. Western blot analysis of ERCC1 in chromatin fraction of Xpf +/– and Xpf∆Nuc/– clones. H3: Histone H3 loading control. Note presence of ERCC1 signal in in-frame deleted Xpf∆Nuc/– clone #1, and absence of ERCC1 signal in frame-shifted Xpf∆Nuc/– clones #2, 11 and 13 k. Cell cycle distribution of Xpf+/– clone #6 and Xpf∆Nuc/– clone #1, either untreated or treated with 20, 30, 40 or 50 ng/mL of MMC. Data shows mean values of three biologically independent replicates, n = 3. P-value: **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars: s.e.m. Untreated samples were compared using the unpaired Student’s t-test; MMC-treated groups were compared by one-way ANOVA. l and m. Tus/Ter-induced LTGC (l) and I-SceI induced LTGC (m) in Xpf+/– and Xpf∆Nuc/– clones. Data shows mean values of six biologically independent replicates, n = 6. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. P-value: ****p < 0.0001.
