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. Author manuscript; available in PMC: 2011 Jul 28.
Published in final edited form as: Oncogene. 2010 Jan 11;29(11):1702–1716. doi: 10.1038/onc.2009.464

Figure 5.

Figure 5

Effect of JNK1/2 downregulation on progression of G2 cells into mitosis and on endoreplication. (a) JNK1/2 downregulation by specific siRNAs. HCT116 cells transfected with a combination of JNK1- and JNK2-specific siRNAs (two siRNAs each for JNK1 and JNK2) or control siRNA for 48 h were synchronized with nocodazole for 6 h, released for 3 h and then synchronized with thymidine. Nocodazole (NOC) was added at 1 h after release. Extracts obtained from cells at the indicated times after release from thymidine synchrony were immunoblotted with antibodies recognizing JNK1/2 and phosphohistone H3. Similarly, immunoblotted extracts from control siRNA-transfected cells treated with 20 μM SP600125 (SP) at 4 h after release from thymidine block is shown as a control. (b) Progression of cells into mitosis after JNK1/2 downregulation. Cells from (a) were harvested at the indicated times, stained with PI and MPM2 and examined by flow cytometry. Numbers in DNA content plots are percentage of population that is 8N. Numbers in MPM2 dot plots are percentage of the 4N population that is in mitosis. Cells transfected with control siRNA and treated with SP600125 exhibited both a significant suppression of mitotic entry and an increase in endoreplication. (c) Downregulation of JNK1/2 by specific siRNAs suppresses JNK activity. Extracts obtained from HCT116 cells transfected with a combination of JNK1- and JNK2-specific siRNA or control siRNA, synchronized with thymidine and released as in (a) were immunoblotted with antibodies recognizing JNK1/2 and its phospho-c-Jun substrate. (d) SP600125 treatment of downregulated JNK1/2 cells results in the generation of 8N population. HCT116 cells transfected with a combination of JNK1- and JNK2-specific siRNA or control siRNA as in (a) were synchronized with thymidine. Cells were treated with 20 μM SP600125 at 4 h after release from thymidine block. Nocodazole was added at 1 h after release. Cells harvested at 48 h were stained with PI and examined by flow cytometry. Numbers in DNA content plots are percentage of population that is 8N.