Kif15 is essential for K5I-resistance in HeLa cells. (A) STLC-resistant colonies emerge from HeLa cell populations, but not from KIF15Δ cell populations. Schematic of experimental design (top). Images of 10-cm dishes seeded with HeLa or KIF15Δ cells, treated with STLC for 28 d, and stained with crystal violet (bottom). Bar, 2 cm. See also Fig. S5. (B) Quantitation of A, with the addition of AZD4877 and Filanesib. Values represent the mean number of colonies detected in 10-cm plates seeded with HeLa (purple) or KIF15Δ (gray) cells and treated with STLC, AZD4877, or Filanesib as indicated for 28 d. Note that no colonies were detected from KIF15Δ cell populations under any scenario. Error bars, ± SEM; n = 3. (C) EGFP-Kif15 levels in transgenic cell lines are under control of doxycycline induction. Western blot of WCLs prepared from indicated cell types probed with antibodies targeting Kif15 (αKif15) and tubulin (DM1α). Numbers indicate days of induction with doxycycline. Note that the lower Kif15 species represents endogenous protein and the higher represents exogenous EGFP-Kif15. (D) EGFP-Kif15 expression rescues K5I resistance in the KIF15Δ cell line. Schematic of experimental design (top). Images of 10-cm dishes seeded with the indicated cell type, induced with doxycycline for 1 d, treated with STLC for 6 d, and stained with crystal violet (bottom). Insets (stippled boxes) are enlarged to show the presence of cell clusters in the HeLa-EGFP line and lack thereof in the KIF15Δ cell line. Bars: 2 cm; (inset) 0.5 cm.