Zen2010 program

For immunofluorescence observation, HeLa cells were seeded on 24-well microplates at 2 or 4 × 10⁴ cells/mL and cultured for 24 h. The cells were further cultured with DMSO, nocodazole (200 nM), BI2536 (200 nM), and KBJK557 (200 μM) for 18 h. The medium in the microplate wells was then removed and cells were fixed with 4% formaldehyde in PBS for 15 min and then permeabilized for 5 min with 0.1% NP-40 in PBS. The cells were washed with PBS containing 1% bovine serum albumin (PBS-1% BSA) and then treated with 5% bovine serum albumin for 20 min. The cells in each well were treated with anti-Plk1 (mouse) and anti-CREST (human) antibody in PBS-0.1% BSA and then placed in a humidified atmosphere at room temperature and incubated for 1.5 h. After being washed with PBS-0.1% BSA, treated wells were incubated with Alexa Fluor 488-conjugated human anti-IgG antibody and Texas Red-conjugated mouse anti-IgG antibody in PBS-0.1% BSA and then incubated at room temperature for 60 min. After being washed with PBS-0.1% BSA, the dish wells were overlaid with DAPI for 10 min at room temperature and then washed with PBS-0.1% BSA. Fluorescence was photographed with an inverted Zeiss 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). The signal intensity was measured by the ZEN2010 program (Carl Zeiss, Oberkochen, Germany).