Fig. 4: G4-stabilizing ECCs inhibit FANCJ unwinding G4, but not DNA2-mediated cleavage of G4 in vitro.

a, b Representative primer extension from three replicates by Polδ (20 nM) on the non-G4 or G4 template in the presence of increasing concentrations of G4-stabilizing ECCs PIPER (0.2, 0.5, 1 µM) (a) and capreomycin (5, 10, 50, 100 µM). b The top shows the diagram of the Polδ (20 nM)-catalyzed primer extension on a DNA template without or with a G4-forming sequence. The primer was labeled with FAM on the 5’ end. The bottom shows the representative denaturing-PAGE image of the primer extension assay; c, d Representative primer extension from three replicates on the G4 template in the absence or presence of 5 nM FANCJ with or without 0.2, 0.5, and 1 μM PIPER (c) or 10, 50, and 100 μM capreomycin (d); e, f Representative blot from three replicates showing the cleavage of a FAM³²P-labeled G4 substrate (50 nM) with DNA2 (16 nM) and MutSα (50 nM) in the absence or presence of increasing concentrations of PIPER (0.5, 1, 2, 5, 10 µM) (e) or capreomycin (1, 10, 50, 100 µM) (f); g, h G4 immunofluorescence staining in WT, DNA2^+/−, and MSH2^−/− MEFs. g The representative AIRYSCAN confocal microscopy images of G4s, and (h) shows the quantification of G4 in WT, DNA2^+/−, or MSH2^−/− MEFs without or with treatment with G4 stabilizers PIPER or capreomycin (n = 45, 47, 51, 44, 50, 50, 41, 50, 48 cells). Results are from at least 3 biological replicates. Red lines indicate median. All p-values were calculated using a two-tailed Student’s t-test (WT NT vs. WT Capreo p < 0.0001; WT NT vs. WT PIPER p < 0.0001; WT Capreo vs. DNA2+/− Capreo p = 0.021; WT PIPER vs. DNA2+/− PIPER p = 0.005; WT PIPER vs. MSH2^−/− PIPER p = 0.005). Scale bar = 5 μm. Source data are provided as a Source data file. Created in BioRender. Zhou, T. (2025) https://BioRender.com/d58fiu1.