Figure 3.

Prediction of the disruption of the interaction between microtubules and αTAT1 by microtubule acetylation inhibitors. (A) Predicted molecular docking models between acetylation inhibitors and the K40 residue in α-tubulin. Chemical-protein interactions and residues in the images were visualized using PyMOL program, and the binding affinity was obtained from GalaxyWEB. Hydrogen bonds are indicated by dashed red lines, and pale cyan sticks represent residues interacting with chemical compounds in α-tubulin. (B) Overexpressed αTAT1 (red), α-tubulin (green), and nucleus (blue) in MDA-MB-231 cells were detected by immunofluorescence staining. Scale bar, 50 µm. (C) DsRed-αTAT1-expressed MDA-MB-231 cell lysates were analyzed by western blotting. (D) Microtubules purified from αTAT1 KO MDA-MB-231 cells were incubated with His-αTAT1, acetyl-CoA, and one of the inhibitors (GM-90257, GM-90568, and GM-90631; 0.5 and 1 mM) or DMSO as a control for 30 min at 37 °C. (E) In vitro microtubule polymerization assay. DMSO was used as a vehicle control, and all reagents including paclitaxel, nocodazole, GM-90257, and GM-90631 were used at the same concentration of 1 µM.