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. 2012 Jul 19;2:516. doi: 10.1038/srep00516

Figure 2. JNK is required for the maintenance of self-renewal capacity of stem-like glioblastoma cells.

Figure 2

(a) Stem-like glioblastoma cells cultured in the presence of SP600125 (40 μM, 7 days for TGS01; 20 μM, 3 days for GS-Y01) were subjected after washout of the inhibitor to serial (i.e., primary to tertiary) sphere formation assays in the absence of SP600125. Top, phase-contrast micrographs of the primary spheres (scale bars, 200 μm). Bottom, number of tumourspheres formed (mean ± s.d. from 3 experiments). *P < 0.05. (b–d) TGS01 and GS-Y01 cells were cultured in the presence of the indicated concentrations of SP600125 for 7 and 3 days, respectively. Cells were then subjected to immunoblot analysis for the expression of phospho-c-Jun (b) and stem cell and differentiation markers (c) and to immunofluorescence staining (green) of nestin, GFAP, and βIII-tubulin (d). In (d), nuclei were counterstained with Hoechst 33342 (blue), and the values in the graph represent mean ± s.d. from 3 experiments. Scale bars, 100 μm. *P < 0.05. (e–f) TGS01 and GS-Y01 cells were transiently transfected with siRNAs against JNK1 or JNK2, or with a control siRNA. Cells were then subjected to serial sphere formation assays (e, values represent mean ± s.d. from 3 experiments)) or to immunoblot analysis to examine the JNK signalling pathway (3 days after transfection) and stem cell/differentiation status (7 and 3 days after transfection for TGS01 and GS-Y01, respectively) (f). *P < 0.05.