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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Nat Cell Biol. 2010 Jun 27;12(7):686–695. doi: 10.1038/ncb2071

Figure 5. Hyperactivation of JNK during unperturbed cell cycle induces aberrant microtubular and chromosomal structures and a prometaphase-like arrest in cells.

Figure 5

(A) Immunofluorescence microscopy performed in HFF-1 cells. Upper left panels depicts normal spindles in cells expressing JNK2 wild-type. Bottom left panels shows cells arrested in early mitosis and abnormal microtubular structures seen upon expression of JNK2ΔKEN. Tubulin is visualized in red and DNA in blue. Graphs on the right panels correspond to the G2/M (mitosis) arrest quantification observed by microscopy in HFF-1 cells expressing JNK2ΔKEN (n = 900 cells counted) versus JNK2 wild-type (n = 1200 cells counted) and the penetrance of the aberrant microtubular structures found in the cells arrested in the mitosis-like state. (B) Flow cytometry cell cycle analyses performed in HFF-1 cells overexpressing the indicated constructs under the stated treatments (noc: nocodazole −18 hours), for a representative experiment used to perform the microscopy depicted in (A). The percentage of cells in G1, S, and G2/M (a mixed population of G2 and mitosis) phases of the cell cycle are included. Experiments were repeated at least three times. (C) Top panels, captions taken from live imaging movies using either HeLa cells stably transfected with GFP-H2B or HFF-1 cells after overexpression of the indicated JNKs (for 24 h). Bottom panel, quantification of the prometaphase-like arrest induced by JNK2ΔKEN expression (for 24 h) in HeLa and HFF-1 cells. (D) Quantification of the percentage of HFF-1 cells in prometaphase-like arrest (as detected by immunofluorescence analysis after 24 h) under the conditions indicated. APF refers to a kinase-dead version of JNK2 (Thr183Ala, Tyr185Phe mutant)32. [i]-n corresponds to three different concentrations (n=1–3) of JNK inhibitor VII (100 nM, 0.5 µM, and 1 µM) used for 16 h. (E) Immunofluorescence analysis of mitotic spindles in HeLa transfected (for 48 h) with JNK2 wild-type (wt) or KEN-deleted mutant (JNK2-ΔKEN). Tubulin is shown in green and DNA (chromosomal) staining with DAPI in blue.