3a, and data not shown). 9 and 10 hours post-release from the second thymidine arrest. (d) Stable HeLa cell lines transduced with empty vector (lanes 1C4) or an siRNA-resistant form of Cdt1 (Cdt1res lanes 5 and 6) were synchronized as in b and transfected with either control siRNA targeting GFP (lanes 1 and 2) or with siRNA (lanes 3C6). Cells were released from early S phase into nocodazole for 10 hr and then either harvested (0 hr) or released for 2 hours into G1 phase, and Cdt1 protein levels were analysed by immunoblotting. (e) Flow cytometric analysis of DNA content of cells in d. To eliminate the possibility that a mitotic phenotype simply reflected replication errors in the preceding S phase, we took advantage of the fact that Cdt1 is actively degraded during S phase after its G1 origin licensing role is complete17 (Fig. 1b). We synchronized cells in early S phase, at which time origins are fully licensed, and released them into medium containing control siRNA siRNA. As expected, control cells had very B2M low amounts of Cdt1 in S phase, and Cdt1 re-accumulated in G2 (Fig. 1c, lanes 1C5). Synchronized cells treated with siRNA could not re-accumulate Cdt1 in G2 however (Fig. 1c, lanes 7C10). (Cdt1 is phosphorylated in PF-06447475 G2 by stress PF-06447475 MAP kinases17, 18) Cdt1 depletion during S phase caused no delay in S phase progression (Fig. S1a and S1b), and by 9 hours after release both control and Cdt1-depleted cells exhibited normal chromosome condensation and/or nuclear envelope breakdown (e.g. Figure 2a and data not shown). This unique experimental approach PF-06447475 allowed us to generate cells that underwent a normal G1 and S phase but lacked Cdt1 during G2 and M phase. Open in a separate window Figure 2 G2-specific Cdt1 inhibition induces mitotic arrest(a) HeLa cells synchronized in early S phase were released into control (luciferase) or siRNA for either 9 hrs (top and middle panels) or 10 hrs (bottom panels) followed by fixation and staining with DAPI to label chromosomes (blue), anti-tubulin antibody to label MTs (red), and anti-Knl1 antibody to label kinetochores (green). (b) Quantification of the results at 10 hr in a by mitotic stage; n = 1500 cells. (cCe) HeLa cells stably expressing GFP-histone H2B were injected with control buffer (c, n=15) or anti-Cdt1 antibody (d, n=27). Selected frames of GFP-histone (top panels) and phase contrast images (bottom panels) are shown. (e) Quantification of the results from the microinjection experiments in c and d. Scale bars = 5 m. See also Supplementary Movies S1CS4. We then tested the ability of Cdt1-depleted cells to transit mitosis to G1 after a nocodazole arrest and release (Fig. 1d and 1e). Control cells completed cell division, but Cdt1-depleted cells remained arrested with G2 DNA content (Fig. 1e, si-control si-cultures PF-06447475 to deplete either Cdt1 or another licensing protein, Orc6, resulted in the accumulation of phospho-H2AX-positive foci, presumably from incomplete replication and fork collapse (Figure S1e). The majority of the synchronized Cdt1-depleted cells arrested just prior to metaphase with most chromosomes positioned near the spindle equator. A smaller fraction of cells arrested in prometaphase (Fig. 2a and 2b; Fig. S2a and S2b). In addition to Cdt1 depletion in synchronized cells, we also microinjected purified.