Supplemental Information Supplementary Figures 1-6

Supplemental 1 A. T47D SUM149PT MCF7 HCC1937 HTB126 HCC1428 HCC1143 SUM190PT MDAMB231 HCC38 HMEC50 KIF2C KIF2A ERK1/2 B. C. EBSS 0 1 2 3 4 0 1 2 3 4 HBEC3KT HBEC3KTRL53 EBSS 0 1 2 3 4 0 1 2 3 4 HBEC3KT HBEC3KTRL53 S1. Expression of kinesin 13 family proteins in cancers and HBEC3KTRL53. (A) KIF2A and KIF2C expression in breast cells T47D, SUM149PT, MCF7, HCC1937, HTB126, HCC1428, HCC1143, SUM190PT, MDA-MB-231, HCC38, HMEC50. (B) Microarray profile of tumor and normal panel cell lines for KIF2B with p value of 0.0006 by two-tailed t test. The signals with KIF2B probes are near the limit of detection. The mean difference is in the range of 10%. In spite of the small p-value, the difference may not be significant. (C) Quantitation of relative KIF2A and KIF2C expression in HBEC3KT and HBEC3KTRL53 from the blots in Fig. 1C as a function of time (0-4 h) in EBSS.

Supplemental 2 HBEC30KT phalloidin α-tubulin merge HBEC3KT phalloidin α-tubulin merge  S2. Cell morphology. (A) HBEC30KT (B) HBEC3KT were immunostained with phalloidin (green), α-tubulin (red) and DAPI (blue) and processed in ImageJ.

α-tubulin HBEC3KTRL53 siControl siKIF2A siKIF2C Supplemental 3 S3. Loss of KIF2A and KIF2C have an effect on cellular morphology. α-tubulin was detected in HBEC3KTRL53 after knockdown of KIF2A or KIF2C.

Supplemental 4 D. E. F. A. B. C. DMSO LY294002 PD0325901 HBEC3KT53 KIF2C ERK1/2 KIF2A DMSO LY294002 HBEC3KT53 HBEC3KTRL53 C. pERK1/2 PD0325901

S4. KIF2B gene expression after MEK-ERK pathway inhibition S4. KIF2B gene expression after MEK-ERK pathway inhibition. (A) HBEC3KT and HBEC3KTRL53 cells were treated with 100 nM PD0325901 for 48 hours. Cell viability was determined using the CellTiterBlue® Cell Viability Assay, according to manufacturer’s protocol. (B) HBEC3KT53 treated with 10 μM LY294002 or DMSO and HBEC3KTRL53 treated with DMSO were compared by immunoblotting to assess KIF2C and KIF2A expression. (C) HBEC3KT53 were treated with 100 nM PD0325901 followed by immunoblotting with designated antibodies. RNA was isolated from (D) HBEC3KT, (E) HBEC3KT53 and (F) HBEC3KTRL53 cells were treated for 48 hours with 1 μM PD0325901 followed by qPCR to measure of KIF2B gene expression. Three different experiments were analyzed by an unpaired t-test resulting in p-values (D) 0.2029 (E) 0.2914 (F) 0.0013.

Supplemental 5 S5. Effect of expression of constitutively active MEK1 on KIF2C expression. Active MEK1 (R4FMEK) was expressed overnight in HBEC30KT and KIF2C was immunoblotted as in Fig. 1.

Supplemental 6 Control Taxol HBEC3KTRL53 S6. Taxol arrests HBEC3KTRL53 cells in mitosis. HBEC3KTRL53 cells were treated for 12 hours with 200 nM of Taxol followed by DNA content analysis as described in Fig. 6A.