FIGURE 2:

Munesib-1 and Fift-IN potently inhibit Kif15 both in vitro and in cells. (A) Chemical structures of Munesib-1 (top) and Fift-IN (bottom). (B) Representative montage of a fluorescently labeled stabilized microtubule in gliding assay utilizing KIF15-N700 treated with either DMSO (left) or 24 µM Munesib-1 (right). Time of each frame is indicated on the left in seconds. Scale bar, 5 µm. (C) CRC generated from ATPase assays with Munesib-1 over a range of eight concentrations from 0.1 to 30 μM. Each concentration was repeated for a total n = 3–9. Error bars show ± SEM. (D) CRC generated from ATPase assays with Fift-IN over a range of eight concentrations from 0.3 to 300 μM. Each concentration was repeated for a total n = 6–9. Error bars show ± SEM. (E) CRC generated from microtubule gliding assays with Munesib-1 over a range of eight concentrations from 10 nM to 30 µM. Each concentration was repeated in triplicate, n ≥ 50 for each replicate. Error bars show ± SEM. (F) CRC generated from gliding assays with Fift-IN over a range of eight concentrations from 10 nM to 100 µM. Each concentration was repeated in triplicate, n ≥ 50 for each replicate. Error bars show ± SEM. (G) Representative montage of a microtubule from a washout assay with the addition of either DMSO (left), 24 µM Munesib-1 (middle), or 100 µM Fift-IN (right). Microtubule motility is shown before drug was added (“Pre Wash-in,” top), after drug was added (“Wash-in,” middle), and after drug was washed out (“Wash-out,” bottom). Time elapsed from the first frame of each phase is indicated on the left in seconds. Scale bar, 5 µm. (H) Quantification of washout experiment showing % inhibition of gliding velocity of KIF15 after addition of drug (“Wash-in,” solid bars) and after washout of drug (“Wash-out,” hatched bars) for the addition and washout of DMSO, 24 µM Munesib-1, or 100 µM Fift-IN. Each compound was tested in triplicate, n ≥ 20 for each replicate. Error bars show ± SEM. Statistical results are shown for an unpaired t test; ns, no significance; ****P < 0.0001. (I) Max intensity z-projections of RPE-1 cells (left) and KIRC-1 cells (right) treated with either DMSO, 50 µM Munesib-1, or 25 µM Fift-IN. Cells were stained with antibodies targeting KIF15 (red) and tubulin (green) and were counterstained with Hoechst 33342 (blue). Scale bar, 5 µm. (J) Quantification of preanaphase spindles in either monopolar or bipolar states in RPE-1 (left) or KIRC-1 (right) cells treated with increasing concentrations of Munesib-1. Concentration is indicated on the bottom of each bar in µM. Each concentration was tested in triplicate, n = 100 for each replicate. Error bars show ± SEM. (K) Quantification of preanaphase spindles in either monopolar or bipolar states in RPE-1 (left) or KIRC-1 (right) cells treated with increasing concentrations of Fift-IN. Concentration is indicated on the bottom of each bar in µM. Each concentration was tested in triplicate, n = 100 for each replicate. Error bars show ± SEM.