Fig. 1
C28 induces the accumulation of mitotic cells and disrupts microtubule dynamics. a Chemical structure of C28. b Synchronized MCF7, T47D, MDA-MB-231 and MCF12A cell lines were analysed by flow cytometry (FACS) following treatment with increasing concentrations (0, 1, 5 and 10 μM) of C28 for 48 h. Percentages of cells in G0/G1, S and G2/M phase are indicated. Results are expressed as mean ± SEM. The experiment was performed two times. c MCF7, T47D, MDA-MB-231 and MCF12A cell lines were treated with increasing concentrations (0, 1, 5 and 10 μM) of C28 for 48 h and mitotic cells were counted. The mitotic index is represented as the percentage of mitotic cells over the total number of cells counted. The experiment was performed two times. ANOVA statistic test was performed using Prism 8 software. Results are expressed as mean ± SEM; * P < 0.05, ** P < 0.01. d Western blotting analysis of the phospho-histone H3 (Ser10) mitotic marker in MCF7, T47D, MDA-MB-231, MCF12A cell lines following treatment with increasing concentrations of C28 for 48 h. GADPH was used as loading control. Values represent the average of three experiments. e Western blotting analysis of the phospho-histone H3 (Ser10) mitotic marker in MCF7, T47D, MDA-MB-231 cell lines following siRNA silencing of LMTK3 for 72 h. GADPH was used as loading control. Values represent the average of three experiments. f MCF7, T47D and MDA-MB-231 cell lines were treated with increasing concentrations (0, 1, 5 and 10 μM) of C28 for 48 h. Cells were centrifuged to separate the insoluble (polymerized) and soluble (un-polymerized) tubulin, and fractions were analysed by western blotting. Colchicine (50 nM) or paclitaxel (50 nM) served as positive or negative control respectively. Values represent the ratio between the insoluble and soluble fractions and are the average of three experiments. g In vitro polymerization of bovine purified tubulin following incubation with increasing concentrations (0, 1, 5, 10 and 20 μM) of C28. Nocodazole (10 μM) or paclitaxel (10 μM) served as positive or negative controls respectively. The optical density (OD) was measured at 350 nm. h Molecular model of C28 bound to the colchicine site of tubulin in comparison with the X-ray crystal structure of tubulin complex with nocodazole (PDB ID: 5ca1) (29). The subunits α and β of tubulin are illustrated as purple and blue ribbons, respectively. Nocodazole (NZO) is shown with green C atoms, whereas C28 with orange C; all other atoms are coloured blue for N, red for O and pink for F. Potential hydrogen-bonding interactions between the ligands and key residues of tubulin are indicated with dashed lines. i Dose-dependent quenching of the intrinsic fluorescence of purified tubulin upon C28 binding. Fluorescence spectra of free tubulin 2 μΜ in PIPES buffer pH 6.9 (black line) and with increasing concentrations of C28 as indicated in the legend. Fluorescence was monitored at 25 °C in the range of 310 to 420 nm by excitation at 290 nm. Inset, is the change in the fluorescence intensity of tubulin heterodimers (ΔF) as a function of C28 concentration, from which the apparent dissociation constant (Kd) was estimated to be 73 ± 15 μΜ by fitting to the equation described in the methods