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. 2022 Nov 4;13:6664. doi: 10.1038/s41467-022-34349-8

Fig. 6. SLF2 and SMC5 patient cells exhibit S-phase associated DNA damage.

Fig. 6

a Percentage of cells positive for EdU staining with >10 53BP1 foci in SLF2 and SMC5 mutant fibroblast cell lines infected with lentiviruses encoding WT SLF2, WT SMC5, or an empty vector. A minimum of 900 EdU positive cells across 3 independent experiments were counted. b SLF2 and SMC5 patient fibroblast cell lines were pulsed with 10 μM EdU for 45 min, fixed, and mitotic DNA synthesis was visualized by mitotic EdU incorporation following labeling with click chemistry. The percentage of mitotic cells with EdU foci was quantified. A minimum of 300 mitotic cells were counted. n = 3 independent experiments. c Immunofluorescent microscopy analysis to quantify the percentage of G1-phase cells (CENPF negative cells) with >3 53BP1 bodies in WT SLF2, WT SMC5, or an empty vector expressing SLF2 and SMC5 patient fibroblasts. n = 3 independent experiments. A minimum of 750 G1-phase cells were counted. d Levels of micronuclei in cells from (c). n = 3 independent experiments. A minimum of 2500 cells were counted. e Levels of micronuclei in U-2 OS SLF2 CRISPR HM cells infected with lentiviruses encoding WT SLF2 or an empty vector. n = 3 independent experiments. A minimum of 1700 cells were counted. f, g Quantification of the average number of chromosomal aberrations per metaphase (which includes chromatid/chromosome gaps, breaks, fragments and chromosomes radials) in WT, SLF2 patient (f), or SMC5 patient LCLs (g). n = 3 independent experiments. A minimum of 140 metaphases were counted. h Average number of chromosomal aberrations per metaphase (chromatid/chromosome gaps, breaks, fragments and chromosome radials) in SLF2 and SMC5 mutant fibroblast cell lines infected with lentiviruses encoding WT SLF2, WT SMC5, or an empty vector was quantified. n = 3 independent experiments. A minimum of 90 metaphases were counted. i Average number of chromosomal aberrations (chromatid/chromosome gaps, breaks, fragments and chromosome radials) per metaphase in U-2 OS SLF2 CRISPR HM cell lines expressing either WT SLF2 or an empty vector. n = 3 independent experiments. A minimum of 100 metaphases were counted. In all cases, a Student’s t test (two-sided, equal variance) was performed for statistical analysis and error bars denote SEM.