Levels of SOX2 protein were compared in the population as a whole and those undergoing mitosis by immunoblotting. (D) Mitotic shake-off was used to enrich for GNS cells in mitosis (confirmed by enrichment for Cyclin B1 left panel). (C) Following BI 2536 treatment, GNS cells display reduced levels of SOX2 but do not have significantly altered levels of the neural stem cell marker SOX9 or upregulation of the astrocyte marker GFAP. (B) Cell cycle profiles of BI 2536-treated NS and GNS cells by flow cytometry confirm a clear increase in the proportion of cells in G2/M for GNS cells, but not normal NS cells. (A)Dose responses to J101 and BI2536 for three different NS and GNS cell lines, assayed using pHH3 immunocytochemistry 24 h after treatment.
(D) Quantification of data from (C) indicates that cells treated with J101 or BI2536 fail to progress to metaphase.īI 2536, a potent and selective PLK1 inhibitor, disrupts GNS cell proliferation but does not trigger astrocyte differentiation. Photomicrographs show aberrant representative spindles of different morphologies (blue, DAPI green, (α-tubulin red, PHH3). Failure of viable bipolar spindle formation was observed for G7 cells treated with J101 or BI 2536. (C) Immunostaining for spindle protein (α-tubulin) and pHH3 confirms that arrest at prometaphase is associated with aberrant spindle formation. (B) Western immunoblot for PLK1 and phospho-T210, and PLK2 in a panel of NS and GNS treated with DMSO, J101 or the PLK1 inhibitor BI2536. However, we also noted a suppression of levels of active Plk1 (shown in bold). Several kinases (bold) were affected including downstream components of the PDGFR signalling pathway. Significant hits are shown and their suggested interactions presented following pathway analysis (Ingenuity). (A) 812 (550 pan- and 262 phospho-specific) proteins were assessed before and after drug treatment by antibody microarrays. GNS cells arrest at prometaphase due to loss of Plk1 activity and failure of spindle assembly. Greater sensitivity of GNS cells is observed across seven different cell lines. (C) Quantification of the ratio between pHH3 stained and total cell numbers (DAPI) from experiments in panel A. Arrows indicate the fragmented nuclear membrane, a feature of prometaphase, in mitotic cells as similar in DMSO controls and following inhibitor treatment. (B) Mitotically arrested cells (G7), were immunostained for pHH3 (white), Lamin B (green) and counterstained with rhodamine-phalloidin to visualise actin (red). (A) Phospho-histone H3 (pHH3) staining (green) for CB660, G179, G166, G144 and G7 after following treatment with DMSO or J101 (100 nM) for 24 h with nuclear counterstaining using DAPI (blue). Immunocytochemistry confirms arrest at prometaphase in response to J101. J101 significantly increased the number of mitoses in G179, but not CB660, without parallel increases in cell number (top panel blue dots), whereas cells with mitotic morphology increased dramatically during the first 1–2 days (bottom panel red dots). (C) Kinetics of change in total cell number and mitosis for G179. (B) Relative number of mitoses scored within 48 h from the start of the experiment for each line and all 160 inhibitors. Erroneously segmented debris or dead cells (green ‘x’) are isolated and discarded from event counts. Interphase cells (blue) and mitoses (red ‘i–iii’). Objects were assigned into different classes/bins.
CELLPROFILER ANALYST EXCLUDING DEAD CELLS SOFTWARE
(A) Segmentation of phase contrast images with high-content analysis software (CellProfiler & CellProfiler Analyst). JNJ-10198402 induces mitotic arrest in GNS cells but not in normal NS cells. (F) Example phase contrast images acquired for G179 and CB660 prior to treatment with J101 (0 h) and 60 h.
CELLPROFILER ANALYST EXCLUDING DEAD CELLS FULL
The full data for the screen are presented in Table S1. 2.2 standard deviations from the average of DMSO controls are shown ( P = 0.01).
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