Cell culture, immunofluorescence assays, and image analysis

TP53^–/– RPE-1 and HeLa cells were cultured in DMEM containing 10% fetal bovine serum (FBS), penicillin, and streptomycin. RPE-1 KIRC-1 and HeLa KIRC-2 cells (Sturgill et al., 2016) were cultured in the same medium with the addition of 10 μM STLC. For immunofluorescence assays, cells were grown on glass coverslips in 6-well dishes and then treated overnight with the desired drug concentrations added to their normal medium. Coverslips were rinsed with 1× phosphate-buffered saline (PBS) and fixed in 100% methanol at –20°C for 10 min and then stained with the following primary antibodies: rabbit anti-KIF15 (Sturgill and Ohi, 2013) at 1:2000 for 1 h and FITC–conjugated mouse anti-α-tubulin (DM1α, Sigma-Aldrich) at 1:500 for 30 min. Alexa-594–conjugated antirabbit secondary antibodies were used at 1:2000 for 45 min. All antibodies were incubated at RT. DNA was counterstained with 5 μg/ml Hoechst 33342 and coverslips were mounted in Prolong Diamond (Thermo Fisher Scientific).

Images were acquired using a 60× 1.4 NA objective (Olympus) on a DeltaVision Elite imaging system (GE Healthcare) equipped with a Cool SnapHQ2 CCD camera (Roper). Optical sections were collected at 200-nM intervals and processed using the ratio deconvolution in SoftWorx (GE Healthcare). Further image processing and analysis was done in ImageJ. Acquisition parameters were kept constant across cell lines and conditions.

To quantify levels of KIF15 and tubulin on the mitotic spindle, an ROI was drawn around the spindle to measure the integrated fluorescence of a single image frame for both the KIF15 and tubulin channels. A smaller oval ROI was drawn outside of the spindle to measure background fluorescence on the KIF15 and tubulin channels. Background intensity corrected for ROI size was subtracted from the spindle intensity of each channel, and corrected intensities were used to calculate the KIF15:tubulin ratio.