Fig. 8. Variants in the RAD18-SLF1/2-SMC5/6 complex compromise the ability of cells to replicate in the presence of stabilized G4 quadruplex structures.

a Left: Average number of segmented chromosomes per metaphase in peripheral blood lymphocytes (PBLs) from SLF2 or SMC5 patients, or an unrelated WT individual. 250 total metaphases were counted from 2 independent blood samples. Middle: Representative images of ‘type 1’ and ‘type 2’ segmented chromosomes. Right: Representative image of a metaphase exhibiting segmented chromosomes from SLF2-P3 PBLs (scale bar: 10 µM). b Representative image of FISH with a centromere-specific probe showing dicentric chromosomes in a metaphase prepared from SLF2-P3 PBLs (scale bar: 10 µM). c Average number of sister chromatid exchanges in metaphase spreads from SLF2 and SMC5 patient-derived LCLs. n = 3 independent experiments. A minimum of 100 metaphases were counted. d Quantification of the IdU:CldU track length ratio in untreated and CX451-treated SLF2 and SMC5 patient fibroblast cells. Cell lines were pulse-labeled first with CldU for 30 min, followed by IdU, with or without 250 nM CX5461, for 30 min. n = 3 independent experiments. A minimum of 250 ongoing fork structures were counted. e Average number of chromosomal aberrations (chromatid/chromosome gaps, breaks, fragments and chromosome radials) per metaphase in SLF2 and SMC5 patient-derived LCLs with and without 24 h exposure to 250 nM CX5461. n = 5 independent experiments. A minimum of 350 metaphases were counted. Student’s t test (two-sided, equal variance) was performed. Error bars denote SEM. f LCL proliferation assay. WT and SLF2 and SMC5 patient-derived LCLs were cultured in increasing concentrations of CX5461 for the time untreated cells took to undergo three population doublings. Cell viability following CX5461 treatment was calculated as a percentage of the number of untreated cells. n = 4 independent experiments. Error bars denote SEM. A two-way ANOVA statistical test was performed. g Quantification of IdU:CldU track length ratio in untreated, pyridostatin-, etoposide- and BMH21-treated SLF2 and SMC5 mutant fibroblast cells. Cell lines were pulse-labeled first with CldU for 30 min, followed by IdU with or without 1µM pyridostatin, 50 nM etoposide or 1 µM BMH21, for 30 min. n = 3 independent experiments. A minimum of 150 ongoing forks were counted. For (c, d, g), red lines denote median values, and a Mann-Whitney rank sum statistical test was performed.