C1 treatment accumulates GSCs in G2/M and triggers subsequent mitotic catastrophe.

<p>A, Proliferation assays on two glioblastoma cell lines (U87 and U251). U87 and U251 cells were treated with 5.7 μM C1 or DMSO. Cells were trypsinized and estimated by counting, in duplicate, after 72 h of treatment. Two different experiments were conducted with similar results. B, Time-Lapse on mitotic U251 cells stably expressing GFP was performed in the absence (DMSO) or in the presence of C1 (at 1 μM). The compound was added to the cell culture just before imaging and then cells were continuously imaged. Three independent experiments were conducted and 10 to 15 fields were followed in each. None of the followed mitotic cells divided in two daughter cells. Representative field is imaged, DNA is in blue and the merge shows GFP and DNA. Several polyploid cells were present in the image. Arrows and arrowheads in upper panels of DMSO and C1- treated cell indicate the same cells through time-lapse. The elapse times are indicated on each photo, in some assays, a zoom of one cell is shown (the red bar represents 5 μm) this cell is present on the former field and labelled with an arrow). Images in bottom panels show DNA only (left) and DNA overlap with GFP (right) after 72-hour treatment with DMSO or C1 (the red bars on each panel represent 20 μm). Arrows in the bottom panel of C1-treated cells indicate mitotic catastrophe by C1 treatment. C, Pictures demonstrate pre-mitotic phase (left panel), mitotic (mid panel) process by full karyokinesis and cytokinesis and after cell division (right panel). MELK expression was determined with immunocytochemistry of GBM1600 cells with anti-MELK antibody (red), chromatin staining with Hoechst stain (blue). Picture of pre-mitosis shows GBM1600 cells highly expressed MELK at pre-mitosis phase (400× magnification). Then GBM1600 cells were treated with 5 μM C1 or control and were subjected to immunocytochemistry 3 days later with anti-MELK and chromatin staining (640× magnification). Data were confirmed by three independent experiments. C1 treated cells are micronucleated at metaphase and followed multinuclear chromatin condensation (mid panel). Right panel show multinuclear asymmetric divided chromatin of C1 treated cell compared with DMSO treated cell. D, Flow cytometric analysis of C1- and DMSO-treated GBM1600 cells with Propidium Iodide at 3 days after treatment shows 62.7% of C1-treated cells resulted in the G2/M arrest, whereas the control cells have 19.3% of the G2/M arrested cells.E, Graph indicating the proportions of live, early apoptotic, and late apoptotic U251 cells with varying doses of C1 or DMSO.