Figure S2.
Validation of midzone kinesin experiments. (A) In the suspended microtubule bridge assay, the z-position of the cargo bead was calibrated by measuring the average intensity of 17 surface-immobilized cargo beads by scanning the microscope objective from −255 to 255 nm relative to the imaging plane. Error bars represent SD. The intensity profile was fitted to a third-order polynomial (red curve, R2 = 0.9995) to obtain the calibration curve. (B–E) Western blots of KIF4A (B), MKLP1 (C), KIF4A and MKLP1 (D), or MKLP2 (E) levels in MCF10A cells transfected with siRNA targeting luciferase (control), KIF4A, MKLP1, or MKLP2 for 48 h, as indicated. GAPDH is shown as a loading control. (F) Live confocal images of the late anaphase phenotype after KIF4A knockdown. In late anaphase, siKIF4A spindles over-elongate and have poorly organized midzone bundles. Scale bars = 5 µm. Brightfield (upper row) and SiR-tubulin (lower row) images represent single z-planes. (G) Spindle length in late anaphase, after the cleavage furrow is visible. Black lines represent mean ± SD. n = 14 siCtrl and 11 siKIF4A spindles. ***P = 1.34 × 10−4, two-sample t test. (H) Live confocal images of the telophase phenotypes after KIF4A or MKLP1 knockdown. The midbody is extended (siKIF4A) and cells have cytokinesis defects (siMKLP1). Scale bars = 5 µm. Brightfield (upper row) and SiR-tubulin (lower row) images represent single z-planes. (I) Helicity of MCF10A anaphase spindles calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 34 and 35 spindles pooled from N = 4 independent experiments each for siCtrl and siMKLP2. n.s., not significant, two-sample t test. (J) Helicity of anaphase spindles in MCF10A cells treated with 0.1% DMSO or 10 µM STLC, calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 42 STLC-treated spindles pooled from N = 4 independent experiments, and the same 54 DMSO-treated spindles shown in Fig. 3 B. n.s., not significant, two-sample t test. Source data are available for this figure: SourceData FS2.

Validation of midzone kinesin experiments. (A) In the suspended microtubule bridge assay, the z-position of the cargo bead was calibrated by measuring the average intensity of 17 surface-immobilized cargo beads by scanning the microscope objective from −255 to 255 nm relative to the imaging plane. Error bars represent SD. The intensity profile was fitted to a third-order polynomial (red curve, R2 = 0.9995) to obtain the calibration curve. (B–E) Western blots of KIF4A (B), MKLP1 (C), KIF4A and MKLP1 (D), or MKLP2 (E) levels in MCF10A cells transfected with siRNA targeting luciferase (control), KIF4A, MKLP1, or MKLP2 for 48 h, as indicated. GAPDH is shown as a loading control. (F) Live confocal images of the late anaphase phenotype after KIF4A knockdown. In late anaphase, siKIF4A spindles over-elongate and have poorly organized midzone bundles. Scale bars = 5 µm. Brightfield (upper row) and SiR-tubulin (lower row) images represent single z-planes. (G) Spindle length in late anaphase, after the cleavage furrow is visible. Black lines represent mean ± SD. n = 14 siCtrl and 11 siKIF4A spindles. ***P = 1.34 × 10−4, two-sample t test. (H) Live confocal images of the telophase phenotypes after KIF4A or MKLP1 knockdown. The midbody is extended (siKIF4A) and cells have cytokinesis defects (siMKLP1). Scale bars = 5 µm. Brightfield (upper row) and SiR-tubulin (lower row) images represent single z-planes. (I) Helicity of MCF10A anaphase spindles calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 34 and 35 spindles pooled from N = 4 independent experiments each for siCtrl and siMKLP2. n.s., not significant, two-sample t test. (J) Helicity of anaphase spindles in MCF10A cells treated with 0.1% DMSO or 10 µM STLC, calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 42 STLC-treated spindles pooled from N = 4 independent experiments, and the same 54 DMSO-treated spindles shown in Fig. 3 B. n.s., not significant, two-sample t test. Source data are available for this figure: SourceData FS2.

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