Fig. 6: Loss of ENSG00000272172.1 increases DNA replication speed and alters replication dynamics.

a Upper panel: Schematic of the DNA fiber assay. OVCAR8 cells were transfected with either control siRNA or siRNA targeting ENSG00000272172.1. After 48 h, cells were treated with 20 µM olaparib for 16 h, followed by sequential labeling with CldU and IdU (30 min each). DNA fibers were then spread and stained with specific antibodies, as described in the Methods. Lower panel: Representative immunofluorescence images of labeled DNA fibers. b Quantification of DNA replication speed in OVCAR8 cells treated as in (a). Replication track length was measured in microns and converted to kilobases per minute using a standard conversion factor. c Fork symmetry analysis of individual replication forks. The ratio of CldU to IdU tract length was calculated to assess fork progression balance. d Left: Schematic of the modified DNA fiber assay. OVCAR8 cells were transfected with siRNAs, and 48 h later, treated with 1 µM ATR inhibitor AZD6738 for 1 hour prior to sequential labeling with CldU and IdU. Right: Quantification of replication speed under ATR-inhibited conditions. e Left: Schematic of origin-to-origin distance measurement. Experimental conditions were identical to (d). Right: Quantification of inter-origin distances, measured as the distance between adjacent replication initiation sites on individual DNA fibers. f Left: Cell cycle distribution of OVCAR8 cells transfected with control or ENSG00000272172.1-targeting siRNA, assessed by flow cytometry following DNA content staining (EDU and DAPI). All fiber measurements represent data from at least three independent experiments with ≥100 fibers analyzed per condition. Data are shown as mean ± SD. Statistical comparisons were performed using unpaired two-tailed Student’s t-tests. Significance is indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).