Figure 2. Spartan localizes to sites of stalled replication. (A) Focus formation of EGFP-Spartan in response to DNA damage or replication stress. U2OS cells stably expressing EGFP-Spartan were treated as indicated. Cells were fixed 3 h after UV (20 J/m2) or 6 h after HU (10 mM) treatment. Where indicated, TX-100 extraction was performed to visualize EGFP-Spartan on chromain. (B) Quantitation of cells containing EGFP-Spartan Foci. Cells were treated as in (A), and percentage of cells containing more than ten EGFP-Spartan foci is presented as mean ± SD (n = 3). (C) Colocalization of EGFP-Spartan with ssDNA. Flag-Spartan was transiently expressed in U2OS cells prelabeled with BrdU for two doubling times. Cells were treated with 10 mM HU for 6 h and coimmunostained with anti-Flag and anti-BrdU in non-denaturing conditions. (D) Colocalization of EGFP-Spartan with RPA. U2OS cells stably expressing EGFP-Spartan were fixed 3 h after UV (20 J/m2) or 6 h after HU (10 mM) treatment and immunostained for RPA. (E) Colocalization of EGFP-Spartan with PCNA. U2OS cells stably expressing EGFP-Spartan were treated with 10 mM HU for 6 h and immunostained for PCNA.