Addition of LB3T to assembled X. laevis nuclei inhibits DNA replication. Nuclei were assembled for 60 min followed by the addition of control buffer (A–C), ∼19 μM LB3T (D–F), or ∼19 μM LB3TRW (G–I) for 30 min followed by 5 μM bio-11-dUTP for 10 min. Nuclei were fixed, pelleted onto poly-l-lysine–coated coverslips, and prepared for immunofluorescence using Hoechst 33342, rhodamine avidin, and anti-LB3. Control nuclei continued to grow after addition of the buffer (A–C) and incorporated bio-11-dUTP (B). Using the same image capture settings, there was less incorporation of bio-11-dUTP after the addition of LB3T (E), and the nuclei appeared to be the same size as those in buffer controls at 60 min. In contrast, LB3T RW had a lesser effect on incorporation of bio-11-dUTP (H). Bars, 5 μm. (J) X. laevis nuclei were assembled for 60 min and different amounts of LB3T were added for 30 min followed by a 10-min incubation in 2 μC [³²P]α-dCTP. The reactions were stopped by the addition of replication sample buffer for 10 min. The samples were run on 0.8% agarose gels and dried under vacuum, and radioactivity was measured. Under these conditions, there was a concentration-dependent inhibition of DNA replication attributable to LB3T. When ∼11 μM LB3T was added, there was an ∼50% decrease, and with ∼19 μM LB3T, replication decreased by ∼92%. (K) We also tested the effect of LB3T RW and determined that at a concentration of ∼19 μM, DNA replication was reduced by ∼50%. Each experiment was repeated three times and the results were averaged.