Figure 5

Fork progression is impaired genome-wide and at telomeres in progerin-expressing cells. (A) Principle of the SMARD experiment. HDF cells expressing NLS-EGFP, EGFP-LA and EGFP-PG were pulsed successively four times with the thymidine analogues EdU and IdU, followed by a chase period. Cells were collected and embedded in agarose in plugs before stretching the DNA fibers onto glass coverslips. Telomeres were visualized by FISH staining and EdU and IdU by immunofluorescence. (B) Examples of fibers obtained by SMARD. A non-replicating telomere corresponds to a detected FISH signal away from the EdU and IdU signals. A replicating telomere corresponds to a FISH signal located at the end of the fiber and in continuity with EdU or IdU signals. (C) Distribution of telomere length measured from DNA fibers performed as in (A). The size of the FISH signal gives an approximation of the corresponding telomere length. Mean with SEM is shown, n = 3. Statistical significance was determined using t-test (****indicates p < 0.0001). (D,E) Quantification of SMARD analyses at the genome-wide level in HDF cells expressing NLS-EGFP, EGFP-LA and EGFP-PG. (D) the graphs represent the replication track lengths (left), and the percentage of stalled replication forks in each replicate (right). (E) Inter Origin Distance (IOD). (F) Quantification of SMARD analyses at telomeres and genomic regions from HDF cells expressing NLS-EGFP, EGFP-LA and EGFP-PG. The graph represents the rate of genomic versus telomere replication. (G) Quantification of SMARD analyses at telomeres from HDF cells expressing NLS-EGFP, EGFP-LA and EGFP-PG. The graphs represent the telomere track length. (D–G) Mean with SEM is shown, n = 3. Statistical significance was determined using t-test (*indicates p < 0.05, **indicates p < 0.01, ****indicates p < 0.0001).