Eight well chamber slide

Immunostaining of KB-VIN was performed as described previously.^{43 (link)} Briefly, KB-VIN cells were seeded on an eight-well chamber slide (Lab-Tech) for 24 h prior to treatment with compounds. Cells were treated with compound for 24 h. Concentrations of reagents were used based on their IC₅₀ used for cell cycle analysis as follows: 0.3 and 3.0 µM of 1, 20 µM 3, and 0.1% DMSO as a control. Cells were fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS) followed by permeabilization with 0.5% Triton X-100 in PBS. Fixed cells were labeled with mouse monoclonal antibody to α-tubulin (B5-1-2, Sigma) and rabbit polyclonal antibody to γ-H2AX (BETHYL Lab.) followed by FITC-conjugated antibody to mouse IgG (Sigma) and Alexa Fluor 546-conjugated antibody to rabbit IgG (Life Technologies). Nuclei were labeled with DAPI (Sigma). Fluorescence-labeled cells were observed using a confocal microscope (Zeiss, LSM700) controlled by ZEN software (Zeiss). Parameters (laser, beam splitter, band-pass filter) for confocal fluorescence imaging were used as follows: track 1 for DAPI (405 nm, 420 nm, 420−1000 nm), track 2 for FITC (488 nm, 544 nm, 490−555 nm), and track 3 for Alexa Fluor 546 (555 nm, 559 nm, 505−600 nm). Confocal images were reconstructed by stacking using ZEN (black edition) software (Zeiss). Final images were prepared by Photoshop CS6 (Adobe).