A412 1

After cells had been seeded onto 12 mm glass coverslips in 24-well plates for 24 hours endogenous KIF18A was depleted as described above and GFP-KIF18A was induced with 2 μg/mL doxycycline. 24 hours after knockdown and induction cells were fixed in −20°C methanol (Fisher Scientific, A412-1) for 3 minutes on ice and stained for rabbit KIF18A 1:100 (Bethyl, A301-080A), guinea Pig CENP-C 1:250 (MBL, PD030), and mouse gamma-tubulin 1:500 (Sigma Aldrich, T5326). All primary antibodies were incubated for 1 hour at room temperature, shaking. Twenty random mitotic cells were imaged for each condition per replicate, and expression level was quantified in ImageJ/Fiji by drawing an ROI around the mitotic spindle and measuring the KIF18A signal within that ROI. Background signal was determined by moving the same ROI to an area with no cells and measuring the GFP signal in that space. The mean background signal for each image was subsequently subtracted from the mean spindle KIF18A signal. All values were then normalized to the mean control background subtracted KIF18A intensity. Median and 95% confidence interval are reported for three individual biological replicates.

After cells had been seeded onto 12-mm glass coverslips in 24-well plates for 24 h, endogenous KIF18A was depleted, as described above, and GFP-KIF18A was induced with 2 μg/mL doxycycline. Twenty-four hours after knockdown and induction, cells were fixed in −20°C methanol (Fisher Scientific, A412-1) for 3 min on ice and stained for rabbit KIF18A 1:100 (Bethyl, A301-080A), guinea pig CENP-C 1:250 (MBL, PD030), and mouse gamma-tubulin 1:500 (Sigma-Aldrich, T5326). All primary antibodies were incubated for 1 h at room temperature with shaking. Twenty random mitotic cells were imaged for each condition per replicate, and the expression level was quantified in ImageJ/Fiji by drawing an ROI around the mitotic spindle and measuring the KIF18A signal within that ROI. The background signal was determined by moving the same ROI to an area with no cells and measuring the GFP signal in that space. The mean background signal for each image was subsequently subtracted from the mean spindle KIF18A signal. All values were then normalized to the mean, background-subtracted KIF18A intensity. Median and 95% confidence interval are reported for three individual biological replicates.