Fig. 6

4SC-202 delays mitosis, interferes with spindle formation, and can directly affect tubulin polymerization. a–c HeLa cells expressing a histone H2B-GFP fusion protein were treated with 1 μM 4SC-202 and the indicated FK228 concentrations and analyzed by time-lapse microscopy for 48 h. Detachment and rounding up of the previously adherent cells was regarded as onset of mitosis. a Photo series illustrating typical outcomes following detachment in the absence or presence of 4SC-202. b Time in mitosis was recorded as the duration of detachment of individual cells. The plot depicts singular value distribution, their median and the interquartile ranges. Statistical significance was tested using nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparison post test (***p < 0.001). c The frequency of the events depicted in sub-figure a) was recorded by counting cells (between 56 and 253 events) for the indicated three different FK228 concentrations (each n = 1) and in three independent experiments for 1 μM 4SC (mean values ± SD). Additionally the percentage of sub-G1 cells was determined following propidium iodide staining. d Photo series derived from time-lapse microscopy of HeLa cells expressing a red fluorescent tubulin-RFP in addition to the H2B-GFP fusion protein. Cells were cultured in the absence or presence of 1 μM 4SC-202. e An in vitro tubulin polymerization assay was performed by addition of GTP to a > 99% pure bovine tubulin solution and monitoring of microtubule formation by measuring the OD at 340 nm in the course of time. All indicated substances were added at a concentration of 10 μM as suggested by the manual