HeLa cells were seeded on coverslips and transfected with plasmids. After 48 h, cells were fixed with 4% formaldehyde, and incubated with anti-HA (Santa Cruz Biotechnology) or anti-Myc (Santa Cruz Biotechnology) antibody, followed by secondary goat anti-rabbit antibody coupled to Alexa488 (Invitrogen) and goat anti-mouse antibody coupled to Alexa546 (Invitrogen). The nucleus was stained with DAPI (Invitrogen). Coverslips were mounted and fluorescence was visualized with 40× magnification on a confocal laser scanning microscope (Carl Zeiss, Inc.), and pictures were analyzed with the ZEN 2009 software.
To count cells with mitotic defects, HeLa cells grown on coverslips were transfected with the indicated siRNA and plasmids for subsequent rescue. After fixation and permeabilization, cells were stained with monoclonal anti-β-tubulin-Cy3™ (Sigma) for 2 h, and the nucleus was counterstained with DAPI. Cells with mitotic defects were counted at 40× magnification with a fluorescence microscope (Nikon eclipse TE 2000-U).
To count cells with mitotic defects, HeLa cells grown on coverslips were transfected with the indicated siRNA and plasmids for subsequent rescue. After fixation and permeabilization, cells were stained with monoclonal anti-β-tubulin-Cy3™ (Sigma) for 2 h, and the nucleus was counterstained with DAPI. Cells with mitotic defects were counted at 40× magnification with a fluorescence microscope (Nikon eclipse TE 2000-U).