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. Author manuscript; available in PMC: 2015 May 21.
Published in final edited form as: Nat Commun. 2014 Nov 21;5:5566. doi: 10.1038/ncomms6566

Figure 1. PKM2 Interacts with MLC2 and Is Critical for Cytokinesis.

Figure 1

Immunoblotting (b, d-f, h) and immunofluorescence (a, c, g, i) analyses were performed with the indicated antibodies. Nuclei were stained with DAPI (blue).

(a) U87 cells in cytokinesis were immunostained with the indicated antibodies. Scale bars, 10 μm.

(b) U87/EGFRvIII cells, synchronized by thymidine double block (2 mM), were released for the indicated time periods. Doxycycline (500 ng/ml) was added at the indicated time point to induce PKM2 shRNA expression. MG132 (25 μM) was added at the indicated time point to sustain the cells in metaphase for 6 h.

(c) U87/EGFRvIII cells expressing mCherry-histone H2B (for chromosome staining) were synchronized by thymidine double block. PKM2 shRNA was induced by doxycycline, as described in (b). MG132 was removed after 6 h incubation, followed by imaging analyses using a DeltaVision deconvolution microscope with a 20 × lens in a CO2 environment chamber. Selected time points are shown. Scale bars, 10 μm.

(d) The indicated cells were treated with or without EGF (100 ng/ml) for 24 h.

(e, f) U87 cells (e) or U87 cells expressing Flag-PKM2 (f), which had been synchronized by double thymidine block (2 mM) for 43 h, were unreleased or released for 9 h, followed by MG132 (25 μM) treatment for 1.5 h to arrest cells at metaphase. MG132 was removed for 30 min before cell harvesting. Immunoprecipitates with the indicated antibodies were incubated with or without CIP (10 units) for 30 min at 37°C and were washed with PBS three times. C, cytokinesis; I, interphase (no thymidine release).

(g) Flag-MLC2-expressing U87 cells, synchronized by double thymidine block (2 mM) for 43 h, were released for 10 h (anaphase). Scale bars, 10 μm.

(h) Control or MLC2 shRNA was expressed in U87/EGFRvIII cells.

(i) U87/EGFRvIII cells expressing MLC2 shRNA and mCherry-histone H2B were synchronized. MG132 (25 μM) was added at the indicated time point as shown in (b) and incubated with the cells for 1.5 h to sustain the cells in metaphase. MG132 were then removed, followed by imaging analyses using a DeltaVision deconvolution microscope. Selected time points are shown. Scale bars, 10 μm.