The following antibodies were used in this study: NuMA - rabbit-anti NuMA (gift from

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1 Materials and Methods Antibodies: The following antibodies were used in this study: NuMA - rabbit-anti NuMA (gift from D. A. Compton); Dynein Intermediate Chain (Chemicon, Temecula, CA); Dynein Light Intermediate Chain - pab JH92 made against recombinant dynein LIC-A(21); p150 Glued - mab P41920 (BD Pharmingen, San Diego, CA); Arp1 - mab 45A(30); γ- tubulin - mab GTU88 (Sigma) or rabbit antiserum pab (Sigma, St. Louis, MO) against peptide EEFATEGTDRKDVFFYK; Centrin-2 - mab hcetn2.4(31); HSET - pab anti- HSET (gift from D. A. Compton); Actin - pab A-2066 (Sigma). Cell Culture and transfection: UPCI cell lines were made in the University of Pittsburgh by SMG and all other cell lines were obtained from American Type Culture Collection. UPCI:SCC lines were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2mM L-Glutamine, 0.05mg/ml gentamicin, and 1% MEM non-essential amino acids (all cell culture media/supplements from Gibco BRL, Grand Island, NY unless otherwise noted). Diploid human fibroblast cells (GM03349B) were cultured in MEM supplemented with 15% FBS. JAR cells were cultured in MEM supplemented with 10% FBS. IMR-32 and SK-HEP-1 cells were cultured in MEM supplemented with 10% FBS and 1% MEM non-essential amino acids. Normal oral cells were grown from uvulopalatopharyngoplasty (UP3) tissue samples in KGM-2 media (Bio-Whittaker, East Rutherford, NJ) as described previously (36). HEK293 human embryonic kidney N1E- 115, A431 and Hs766T cells were cultured in DMEM supplemented with 10% FBS. 1

2 MIA-PaCa2 cells were cultured in DMEM supplemented with 10% FBS and 2.5% horse serum. HCT116 and MES-SA cells were cultured in McCoy s 5A medium supplemented with 10% FBS. AGS, PC-3 and A549 cells were cultured in F12K medium supplemented with 10% FBS. All cultures were grown at 37 0 C with 5% CO 2. Cells were seeded on 22mm 2 coverslips (Corning Glass Works, Corning NY) and for sirna transfections were transfected with 1 µg/coverslip of sirna to NuMA using Lipofectamine or Lipofectamine 2000 (both Gibco) following the manufacturer s instructions and then incubated for three days prior to fixation. sirna to GAPDH was used as a negative control. Fluorescently-labeled sirna was prepared using the Silencer labeling kit (Ambion, Austin, TX). Plasmids encoding NuMA (14) and DsRed-tagged CC1 (25, 32) were transfected using Lipofectamine or FuGene6 (Roche Diagnostics, Indianapolis, IN) and fixed hours after transfection. HEK293 cells treated with Colcemid were incubated for hours in 2nM colcemid (Irvine Scientific, Santa Ana, CA) and then released overnight prior to fixation. UP3 cells were cultured in SMEM Ca 2+ free media as described previously(33) Immunofluorescence microscopy: Immunofluorescence was performed as described (6). Briefly, cells were fixed for 5 minutes in -20 C methanol, treated with blocking solution, treated with primary antibodies, washed and then treated with secondary antibodies and 4',6-Diamidino-2- phenylindole (DAPI) which stains chromatin. Samples were scored using a BX-60 microscope (Olympus, Melville, NY). At least 200 cells were scored per condition per experiment or time point, and each experiment was repeated at least four times. 2

3 Western Blot analysis: Whole cell lysates were obtained by harvesting the cells in RIPA buffer (50 mm Tris- HCl, ph 8.0, 150 mm NaCl, 0.5% Na-deoxycholate, 1% NP-40, 0.1% SDS) containing the protease inhibitors leupeptin, pepstatin and PMSF. After centrifugation, cell extracts were analyzed by immunoblotting on PVDF membrane. Blots were probed with primary antibodies overnight at 4 C or for 2 hours at room temperature and then with secondary antibodies coupled to horseradish peroxidase for one hour at room temperature. Immunoreactivity was detected by chemiluminescence using SuperSignal substrate (Pierce, Rockford, IL) as per manufacturer s protocol. 3

4 Table S1. sirna-mediated knockdown of NuMA leads to a loss of multipolarity and restoration of spindle dynein in some cancer cell lines. Cells were scored for frequency of multipolarity in the metaphase population and dynein localization on the spindle before and after sirna knockdown of NuMA. All numbers are % of population, or % of transfected population in sirna-treated cases. The numbers represent total cells, of which approximately 50% are transfected. *In UPCI:SCC078 cells, it was not possible to differentiate transfected cells from the population due to the morphology of the cell population, multipolar Dynein Dynein positive Cell line Tissue source multipolar after sirna positive after sirna NuMAdependent UPCI:SCC103 oral cancer SK-HEP-1 liver cancer UPCI:SCC078* oral cancer NuMAindependent UPCI:SCC070 oral cancer JAR placental cancer IMR-32 brain cancer MES-SA uterine cancer

5 Table S2. Multipolarity in the tested cancer cells arises only in those that exhibit both extra centrosomes and dynein depletion. Cells were scored for frequency of cells with extra centrosomes in the interphase population, dynein localization on the spindle and multipolarity. Spindle localization of dynactin was also determined and all cell lines examined were shown to be >90% positive. All numbers are % of population. Cells were grouped according to the phenotypes seen as follows: cells with I. extra centrosomes and no dynein staining; II. normal centrosomes and no dynein staining; III. extra centrosomes and normal dynein labeling; and IV. normal centrosomes and dynein. Only cells in class I exhibited high (>10%) multipolarity, but further analysis will be required to confirm that this relationship is general one. extra dynein group cell line tissue source centrosomes positive multipolar UPCI:SCC070 oral cancer UPCI:SCC078 oral cancer UPCI:SCC103 oral cancer JAR placental cancer I. IMR-32 brain cancer SK-HEP-1 liver cancer MIA-PaCa 2 pancreas cancer A549 lung cancer MES-SA uterine cancer HCT 116 colon cancer

6 II. A-431 skin cancer PC-3 prostate cancer III. UPCI:SCC114 oral cancer UP3 oral normal IV. GM03349B skin fibroblast HEK293 embryonic kidney normal

7 Fig. S1. NuMA expression levels are decreased in cells treated with sirna. UPCI:SCC103 cells are shown transfected with fluorescently-labeled sirna to NuMA (green). Cells are stained with antibodies to NuMA (red) and with DAPI (blue). Some NuMA persists on spindles in transfected cells, but is considerably depleted when compared to untransfected cells. Fig. S2. sirna knockdown of NuMA has no effect on dynein and dynactin expression levels. Normal human fibroblast cells were transfected with sirna to NuMA and total cell extracts were immunoblotted for NuMA, the p150 Glued subunit of dynactin, and the dynein intermediate chain. Actin was used as a loading control. Note that on some immunoblots NuMA is resolved into two bands, the slower migrating band is believed to be the phosphorylated mitotic isoform (15). Fig. S3. Scoring of extra centrosomes in interphase cells. HEK293 cells were transfected with plasmids expressing hmps1 or treated with Colcemid and stained with antibodies to γ-tubulin. Interphase cells were scored for the presence of extra (>2) γ-tubulin containing centrosomes. Both treatments show an increase in cells with extra centrosomes when compared to untreated cells. Similar results were seen using antibodies to centrin-2 to label centrosomes. Additionally, the oral cancer cell line UPCI:SCC114 showed a high frequency of interphase cells with extra centrosomes, but not multipolar spindles (see Fig. S4). 7

8 Fig. S4. Multipolarity is only seen in cells that have supernumerary centrosomes and depletion of spindle dynein. (A,B) HEK293 cells were transfected with plasmids expressing hmps1 and NuMA or CC1 and scored for spindle polarity and dynein localization to the spindle. (C-E) UPCI:SCC:114 cells were labeled with antibodies to dynein (green) and γ-tubulin (red) and with DAPI (blue) and found to have existing centrosomal amplification (C). For these cells, overexpression of NuMA or CC1 alone led to a substantial increase in multipolar spindles. 8

9 Figure S1

10 NuMA p150 Glued Dynein IC actin Figure S2

11 100 Normal extra centrosomes % interphase cells untreated Colcemid hmps1 ox untreated HEK293 UPCI:SCC114 Figure S3

12 A HEK293 B % metaphase cells untr. hmps1 ox hmps1, NuMA ox bipolar multipolar hmps1, CC1 ox % metaphase cells untr. hmps1 ox hmps1, NuMA ox Dynein+ Dynein- hmps1, CC1 ox C UPCI:SCC114 D % metaphase cells untr. bipolar multipolar NuMA ox CC1 ox E % metaphase cells Dynein+ Dyneinuntr. NuMA ox CC1 ox Figure S4