Figure 4.
TonEBP Promotes Replication Fork Reversal
(A) The TonEBP protein level was determined by western blot analysis. HEK293 cells transfected with scrambled siRNA (S) or TonEBP-targeting siRNA (T). Whole-cell extracts were subjected to western blot analysis.
(B) Representative images of DNA fibers are shown.
(C) Analysis of the replication fork reversal in a DNA combing assay. The iodo-deoxyuridine/chloro-deoxyuridine (IdU/CIdU) ratio was measured in at least 200 well-isolated DNA fibers from three independent experiments; mean ± SD, *p < 0.05, **p < 0.01.
(D and E) U2OS cells transfected with TonEBP-targeting siRNA (T) together with a plasmid expressing tagged Yc1, Yc1 ΔIPT, or Yc1 ΔRHD. U2OS cells irradiated with UV light (60 J/m2) (D) or microirradiated with 405-nm UV light (E) were fixed and immunostained with the anti-ZRANB3 antibody. Right: The percentage of cells with ZRANB3 foci (D) and intensity of UV light-induced ZRANB3 stripes (out of 50 nuclei) (E) expressed as the mean ± SD, n = 3; *p < 0.05, **p < 0.01. Scale bar, 2 μm.