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Fig. 2 | Journal of Experimental & Clinical Cancer Research

Fig. 2

From: Flap endonuclease 1 and DNA-PKcs synergistically participate in stabilizing replication fork to encounter replication stress in glioma cells

Fig. 2

FEN1 Deficiency Inhibit Glioma Cells Proliferation, Induces Increased Replication Fork Degradation in Response to Replication Stress. a Detection of FEN1-RPA interaction was carried out by PLA labeling in M059K cells treated with or without 2 mM HU for 4 h. Representative images are shown. Scale bars, 5 μm. The scatterplot displays quantification of the PLA signals per nucleus from at least 100 cells from three independent experiments. Data are mean ± s.e.m. b M059K cells were transfected with control (siNC) or FEN1 siRNA (siFEN1) for 48 h and then treated with the indicated doses of HU for 4 h or not. Immunofluorescence labeling was performed to detect level of BrdU for ssDNA accumulation analysis. Quantitation of BrdU was presented from three independent replicates. Data are mean ± s.d. c Representative images of γ-H2AX by immunofluorescence labeling are shown. d Quantitation of γ-H2AX was presented from three independent replicates. Data are mean ± s.d. e Schematic of the CldU/IdU pulse-labeling analysis used to investigate nascent strand degradation upon HU treatment in M059K cells transfected with siFEN1 targeting FEN1 for 48 h. Representative images of CldU and IdU replication tracks (top) and scatterplot of IdU/CldU-tract length ratios (bottom) for replication forks are shown. Fiber evaluated from at least 150 events from three independent experiments. Data are mean ± s.e.m. f Schematic of an alternative CldU/IdU pulse-labeling protocol to investigate fork degradation upon HU treatment (2 mM, 4 h) in M059K cells transfected with siNC or siFEN1. Representative images and scatterplots of CldU tract length of individual forks are shown. Fiber evaluated from at least 150 events from three independent experiments. Data are mean ± s.e.m. For PLA experiments, a two-sided Mann–Whitney rank-sum test was used to determine if differences were significant. For immunofluorescence quantification analysis, a two-sided unpaired t test was used to calculate P-values. NS: not significant: P > 0.05

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