A Novel Microtubule Inhibitor 4SC-207 with Anti-Proliferative Activity in Taxane-Resistant Cells
Figure 2
4SC-207 induces mitotic delay/arrest.
Synchronized HeLa wt cells were treated at the end of S phase with 4SC-207, nocodazole (10, 30 and 100nM), or 0.1% DMSO and the first mitotic division was followed by time-lapse microscopy. (A) Scatter plot distributions of the mitotic durations for the different treatments from one representative experiment (for each condition 100 cells were counted and each dot represents one cell that completes mitosis in the indicated time). (B) Quantification of the mitotic durations from three independent experiments. Bars represent means + SEM (standard error of the mean). (C) Cells were categorized into four groups according to their fate after mitotic entry: cells that divided normally (‘normal division’, green), cells that died in mitosis (‘mitotic death’, grey), cells that divided into more than two daughter cells or failed to complete cytokinesis (‘aberrant division’, blue), and cells that arrested in mitosis (‘mitotic arrest’, red). Representative images for each category are displayed (scale bar 5μm). The mean of three independent experiments (100 cells per condition per experiment) + SEM is summarized in the bar chart. (D) HeLa wt cells were first thymidine synchronized then treated with 30nM nocodazole, 100nM 4SC-207 or 0.1% DMSO. 9hrs after thymidine release, cells were fixed and immuno-stained for α-tubulin (green) and DNA (Hoechst, red). Representative images are displayed as individual and merged channels (scale bar 5μm). (E) Quantification of spindle morphology from synchronized HeLa cells stably expressing H2B-mCherry/mEGFP-α-tubulin treated with 0.1% DMSO, 50nM nocodazole or 50nM 4SC-207. Spindles were categorized into three groups: bipolar, ball-like and multi-polar. The mean of three independent experiments + SEM is summarized in the bar chart (the total number of spindles for DMSO, nocodazole and 4SC-207 treated cells were, respectively, 138, 125 and 117). Statistical significance between compound treated and DMSO treated cells in panels (B), (C) and (E) was evaluated using the Student’s t-test and indicated with * (p<0.01 for panel (B) and (E), p<0.001 for panel (C)).