Figure 5.
Figure 5. Generation of the KIF15Δ cell line. (A) Schematic of KIF15 showing target sequence used for CRISPR-mediated genomic editing. Regions encoding the motor domain (blue), MT binding site (green), and autoinhibitory domain (red) are highlighted. Numbers indicate nucleotide position within the ORF. (B) Three distinct INDELs disrupt KIF15 in the KIF15Δ cell line. DNA sequences (left) and the translated protein sequences (right) for WT and KIF15Δ INDELs 1–3 are shown. Bold letters indicate the guide sequence used for CRISPR. Colored letters indicate inserted nucleotides. Underlined codons and asterisks indicate a premature stop. Numbers indicate nucleotide or amino acid position within the KIF15 ORF. See also Fig. S4. (C) Kif15 protein is undetectable in the KIF15Δ line. Western blot of WCLs prepared from HeLa or KIF15Δ cells probed with antibodies targeting Kif15 (αKif15) and tubulin (DM1α). Numbers represent the levels of Kif15 protein in HeLa or KIF15Δ WCLs normalized to the parental HeLa line, averaged over two experiments. Error bars, ± SD; *, P = 0.006 relative to parental HeLa line. (D) Kif15 spindle localization is undetectable in KIF15Δ cells. Maximum intensity z-projections of HeLa or KIF15Δ cells stained with antibodies targeting Kif15 (grayscale and red) and tubulin (green). DNA, blue. LUTs for individual channels are scaled identically. Bar, 10 µm. (E) Quantitation of D. Values represent the fluorescence intensity of Kif15 on individual spindles in HeLa or KIF15Δ cells normalized to the mean such value from HeLa cells, presented as a box-and-whisker plot highlighting the median, 10th, 25th, 75th, and 90th percentiles. n = 20; *, P = 0.004 × 10−9 relative to parental HeLa line. (F) Spindle length is decreased in the KIF15Δ line. Values represent the mean spindle length in HeLa and KIF15Δ cells. Error bars, ± SEM; n = 150 cells from three independent experiments. *, P ≤ 0.01 × 10−29 relative to the parental HeLa line. (G) Mitotic progression is not significantly altered in the KIF15Δ line. Values represent the mean MI of the HeLa and KIF15Δ cell lines. Error bars, ± SEM; n ≥ 1500 cells from three independent experiments. (H) Values represent the mean MPI of the HeLa and KIF15Δ cell lines. Error bars, ± SEM; n = 300 cells from three independent experiments.

Generation of the KIF15Δ cell line. (A) Schematic of KIF15 showing target sequence used for CRISPR-mediated genomic editing. Regions encoding the motor domain (blue), MT binding site (green), and autoinhibitory domain (red) are highlighted. Numbers indicate nucleotide position within the ORF. (B) Three distinct INDELs disrupt KIF15 in the KIF15Δ cell line. DNA sequences (left) and the translated protein sequences (right) for WT and KIF15Δ INDELs 1–3 are shown. Bold letters indicate the guide sequence used for CRISPR. Colored letters indicate inserted nucleotides. Underlined codons and asterisks indicate a premature stop. Numbers indicate nucleotide or amino acid position within the KIF15 ORF. See also Fig. S4. (C) Kif15 protein is undetectable in the KIF15Δ line. Western blot of WCLs prepared from HeLa or KIF15Δ cells probed with antibodies targeting Kif15 (αKif15) and tubulin (DM1α). Numbers represent the levels of Kif15 protein in HeLa or KIF15Δ WCLs normalized to the parental HeLa line, averaged over two experiments. Error bars, ± SD; *, P = 0.006 relative to parental HeLa line. (D) Kif15 spindle localization is undetectable in KIF15Δ cells. Maximum intensity z-projections of HeLa or KIF15Δ cells stained with antibodies targeting Kif15 (grayscale and red) and tubulin (green). DNA, blue. LUTs for individual channels are scaled identically. Bar, 10 µm. (E) Quantitation of D. Values represent the fluorescence intensity of Kif15 on individual spindles in HeLa or KIF15Δ cells normalized to the mean such value from HeLa cells, presented as a box-and-whisker plot highlighting the median, 10th, 25th, 75th, and 90th percentiles. n = 20; *, P = 0.004 × 10−9 relative to parental HeLa line. (F) Spindle length is decreased in the KIF15Δ line. Values represent the mean spindle length in HeLa and KIF15Δ cells. Error bars, ± SEM; n = 150 cells from three independent experiments. *, P ≤ 0.01 × 10−29 relative to the parental HeLa line. (G) Mitotic progression is not significantly altered in the KIF15Δ line. Values represent the mean MI of the HeLa and KIF15Δ cell lines. Error bars, ± SEM; n ≥ 1500 cells from three independent experiments. (H) Values represent the mean MPI of the HeLa and KIF15Δ cell lines. Error bars, ± SEM; n = 300 cells from three independent experiments.

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