Fig. 4

Enhanced interaction of FEN1 and Replisomes and triggered Impaired Fork Progression n with DNA-PKcs Deficiency. a-d Enhanced interaction of FEN1 with MCM2 and MCM5 in M059J cells or M059K transfected with siDNA-PKcs by PLA analysis. e Co-immunoprecipitation revealed elevated interaction of FEN1 and replisomes in DNA-PKcs deficient cells exposed to HU. f Fork degradation was evaluated upon HU treatment in M059K and M059J cells transfected with the siNC or siFEN1 for 48 h. Representative images of CldU and IdU replication tracks and scatterplots of IdU/CldU-tract length ratios for individual replication forks are shown. Fibers evaluated from more than 150 counts from three independent experiments. Data are mean ± s.e.m. g CldU length track assay with HU treatment before IdU label. Representative images of CldU and IdU replication tracks and scatterplots of IdU/CldU-tract length ratios for individual replication forks are shown. Fibers evaluated from more than 150 counts from three independent experiments. Data are mean ± s.e.m. h, i Fork degradation was evaluated in two additional glioma cell lines that were transfected with siFEN1, siDNA-PKcs or combined followed by HU treatment. j, k Schematic of an alternative CldU/IdU pulse-labeling protocol to investigate fork degradation upon HU treatment in M059K and M059J cells transfected with siNC or siFEN1. Quantification of stalled and ongoing forks are assayed. Fibers evaluated from more than 150 counts from three independent experiments. Data are mean ± s.e.m, a two-sided Mann–Whitney rank-sum test was used to determine if differences were significant (a-d, and f-i). A two-sided unpaired t test was used to calculate P-values for stalled and ongoing forks analysis (j, k). NS: not significant: P > 0.05