Fig 2: Oncogene expression combined with FANCA deficiency compromises OFM and FEN1 recruitment following PARPi treatment.a,b, BrdU comet assays were performed on hTERT RPE-1 sgControl or sgFANCA cells with doxycycline-inducible (a) c-Myc (sgControl_Myc or sgFANCA_Myc) or (b) Cyclin E (sgControl_Cyclin E or sgFANCA_Cyclin E) expression, either untreated or treated with Olaparib (10 μM) for 4 hours. Representative comet images (left) and quantification of comet tail moment (right) are shown. Data represent the median ± interquartile range across two biologically independent experiments, analyzed by unpaired two-tailed t-test. ** p<0.01, ***p<0.001, **** p<0.0001. Scale bars, 50 μm. c, Representative IF images of FEN1 and PCNA in hTERT RPE-1 cells with the indicated genotypes under doxycycline induction and either untreated or treated with Olaparib for 4 hours (left). FEN1 foci were quantified and plotted (right). Data shown are mean ± SEM, pooled from three biologically independent experiments, and analyzed by unpaired two-tailed t-test. **p<0.01, **** p<0.0001. Scale bars, 10 μm. d, IF analysis of FEN1 foci in the indicated hTERT RPE-1 genotype cells under doxycycline induction (200 ng/ml) either untreated or treated with HU (250 nM) for 3 hours. Data are mean ± SEM (n= 2 biologically independent experiments), and analyzed by unpaired two-tailed t-test. **p<0.01, **** p<0.0001.

Fig 3: The effect of lentiviral infection on the S1 DNA fiber spreading assay is transient and does not require genome integration.(A) U2OS cells were infected with shLUC-encoding lentiviral particles and subjected to the S1 DNA fiber spreading analysis with and without FEN1 inhibitor seven and ten days after infection. (B) U2OS cells were infected with the same amount of wild type versus integration-deficient (D64V) lentiviral particles encoding YFP. The multiplicity of infection (MOI) was estimated to be between 3 and 30 based on p24-derived viral titers and the assumption that transducing units per ml are 100 to 1000-fold lower than physical units per ml. Four days after infection, cells were subjected to the S1 DNA fiber spreading assay with and without FEN1 inhibitor as described earlier. More than 150 dual-colored fibers were quantified for each biological condition. Three independent experiments were performed showing similar trends. P values were based on Kruskal–Wallis test with Dunnett’s multiple comparisons.

Fig 4: Single-stranded DNA gaps due to FEN1 loss-of-function by lentiviral shRNA infection but not chemical inhibition can be detected by DNA fiber spreading assay.(A) U2OS cells were sequentially labeled with 30 μM CldU for 25 min and 250 μM IdU for 45 min during which time 10 μM FEN1 inhibitor LNT1 was present or absent. Cells were subsequently subjected to the S1 DNA fiber spreading analysis. (B) U2OS cells were infected with lentiviral particles encoding shRNA sequences targeting firefly luciferase (shLUC) or human FEN1 (shFEN1), and subjected to the S1 DNA fiber spreading assay after four days. Western blots for FEN1 and tubulin are shown. More than 150 dual-colored fibers were quantified for each biological condition. Three independent experiments were performed which showed similar trends. P values were based on Kruskal–Wallis test with Dunnett’s multiple comparisons.

Fig 5: Lentiviral exposure enables the detection of lagging strand ssDNA gaps by the S1 DNA spreading assay independently of the vector backbones and transgenes.U2OS cells were infected with lentiviral particles encoding either shLUC in the pLKO.1 vector (A) or YFP in the pFLRu-FH vector (B). Four days after infection, cells were subjected to the S1 DNA spreading assay with and without FEN1 inhibitor and analyzed as in Fig.1. (C) U2OS cells were exposed to conditioned media lacking or containing shLUC lentivirus five days prior to the S1 DNA fiber spreading assay with FEN1 inhibitor. (D) U2OS cells were infected or not with 0.5 μl of purified shLUC lentivirus (MOI of 5) diluted by 1 ml fresh medium and subjected to the S1 DNA fiber spreading assay with FEN1 inhibitor seven days later. More than 150 dual-colored fibers were quantified for each biological condition. Three independent experiments were performed showing similar trends. P values were based on Kruskal–Wallis test with Dunnett’s multiple comparisons.