Fig. 5. The iron depletion-induced cell cycle arrest is independent of checkpoint activation. (A) EJ30 cells were subjected to 24-hour treatments with mimosine or aphidicolin in the absence or presence of 25 µM wortmannin, and stained for DNA (red) and -H2AX (green). (B) Representative cell nuclei from these experiments, stained for RPA. (C) Flow cytometry profiles of HeLa cells treated with mimosine for 28 hours (top); with mimosine only for 24 hours, followed by mimosine plus wortmannin for 4 hours (middle); or with mimosine only for 24 hours, followed by mimosine plus caffeine for 4 hours (bottom). (D) Nuclei from cells subjected to 24-hour treatments with mimosine (mim) or wortmannin (wort) or both (mim/wort) as in A were analysed by in vitro replication reactions. Nuclei were incubated in an elongation buffer (buffer) or in the same buffer supplemented with 100 µg HeLa cell cytosolic extract (cytosol). Percentages of nuclei incorporating labelled nucleotides are shown. (E) Nuclei from cells subjected to 24-hour treatments with caffeine (caff), mimosine (mim) or both (mim/caff) were analysed by in vitro replication reactions as in D.