JNK inhibitors block nocodazole induced Brd4 release and prevent mitotic progression.

<p>A. SP600125 (10 µM), PD98059, U0126 or SB20458 (100 µM) was added to cells 30 min or 2 h prior to nocodazole addition, respectively. The inhibitors were present during the subsequent 4 h of nocodazole treatment (see top for the treatment scheme). Representative immunostaining data are shown below, depicting the localization of endogenous Brd4 and α-tubulin. Of about 200 SP600125 treated cells inspected, more than 90% of cells retained Brd4 on mitotic chromosomes after nocodazole treatment. B. Cells were treated with SP600125 and nocodazole as above and tested for differential salt extraction as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034719#pone-0034719-g001" target="_blank">Figure 1B</a>. C. Extracts from cells treated with or without nocodazole were blotted against total JNK or phosphorylated JNK. α-tubulin was tested as a loading control. The right lane represents samples from mitotic cells collected by mitotic shake-off. D. SP600125 was added to cells that had been treated with nocodazole for 3 h. Cells were incubated for an additional 1 h in the presence of the inhibitor and nocodazole before harvest, and immunostained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034719#pone-0034719-g004" target="_blank">Figure 4A</a>. Of 200 mitotic cells counted, about 85% of cells treated with SP600125 and nocodazole retained Brd4 on chromosomes, while less than 10% of cells treated with nocodazole alone did. E. Cells were incubated with of control or JNKI-1 peptides (5 µM) for 30 min prior to 4 h nocodazole treatment. About 80% of JNKI-1 treated cells did not release Brd4 from chromosomes, while 90% of cells treated with the control peptide released Brd4. F. Cells treated with SP600125 and nocodazole using the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034719#pone-0034719-g004" target="_blank">Figure 4A</a> were allowed to proceed through mitosis for indicated times (min), and stained with Hoechst33324 and cells in anaphase/telophase were counted as dividing cells. The values represent the average of three determinations +/– S.D. * indicates p<0.005.