Figure 3: Some WRN c. 2500C > T homozygous cells display replication fork phenotypes. | Scientific Reports

Figure 3: Some WRN c. 2500C > T homozygous cells display replication fork phenotypes.

From: Homozygosity for the WRN Helicase-Inactivating Variant, R834C, does not confer a Werner syndrome clinical phenotype

Figure 3

Replicating DNA in log phase control and TT homozygous LCLs were labeled with halogenated nucleosides, 5-ethynyldeoxyuridine (EdU) and iododeoxyuridine (IdU) before and after incubation with hydroxyurea (HU), respectively. Cells were lysed, and high molecular weight DNA was isolated and stretched on cover slips using pre-fabricated microchannels. EdU and IdU tracks were stained with fluorescent antibodies and visualized by fluorescent microscopy (Zeiss Axiovert microscope). Track lengths, pre- and post- HU treatment, were measured using AxioVision software. Cumulative ratios of 2nd to 1st labeled segments in two-segment tracks (n = 100–200/sample) were calculated and graphed. A decrease in the rate of DNA chain elongation is manifested as a left shift (decreased IdU/EdU ratios). Controls (black lines): 1405, 1904, 2603, and 3305; Homozygous (grey lines): 1401, 1402, 1903, 2604, and 3304. Open symbols: (−) HU; closed symbols: (+) HU.

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