Figure 2.

Effect of compound 14b on tubulin. (A,B) In vitro tubulin polymerization assay of compound 14b (5 μM), paclitaxel (TAX, 5 μM), colchicine (5 μM) and millepachine (5 μM). (C) Immunofluorescence assay of compound 14b interferes with intracellular microtubules with an LSM 570 laser confocal microscope (Carl Zeiss, Jena, Germany). A549 cells were treated with or without compound 14b (10, 20 and 50 nM), taxol (100 nM) and colchicine (5 μM) for 12 h, and followed by immunofluorescence. The experiments were performed at least three times, and representative images are shown. Scale bars are 10 μm. (D) Superimposition of the docked conformation of the compound 14b on top of the X-ray structure of DAMA-colchicine (PDB code: 1SA0). The backbone of tubulin is shown in ribbon representation (α-tubulin, yellow; β-tubulin, green). (E) Competitive combining capacity of compound 14b and colchicine by the colchicine competition binding assay. The treated concentrations of compound 14b and colchicine were 10, 5, 2.5, 1.25, 0.625 and 0.3125 μM. Additionally, the IC₅₀ values of compound 14b and colchicine when inhibiting the binding of colchicine to tubulin were 1.04 ± 0.02 μM and 1.67 ± 0.05 μM, respectively. The data were shown as mean ± SE (standard error) of at least three independent experiments.