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

Similar documents
Supplemental Information

Supplementary Material

B. ADM: C. D. Apoptosis: 1.68% 2.99% 1.31% Figure.S1,Li et al. number. invaded cells. HuH7 BxPC-3 DLD-1.

SUPPLEMENTARY INFORMATION FIGURE 1 - 1

Description of supplementary material file

RNA was isolated using NucleoSpin RNA II (Macherey-Nagel, Bethlehem, PA) according to the

SUPPLEMENTARY INFORMATION

Supplementary methods

LINGO-1, A TRANSMEMBRANE SIGNALING PROTEIN, INHIBITS OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION THROUGH INTERCELLULAR SELF- INTERACTIONS.

IgG TrueBlot Protocol for Mouse, Rabbit or Goatderived Antibodies - For Research Use Only

Fig. S1 TGF RI inhibitor SB effectively blocks phosphorylation of Smad2 induced by TGF. FET cells were treated with TGF in the presence of

Cell culture and drug treatment. HEK 293 cells were cultured in DMEM (Gibco-BRL)

Apoptosis And Anti-tumor Effect Induced By Mtor Inhibitor And Autophagy Inhibitor In Human Osteosarcoma Cells

Supplementary Materials and Methods

Flow cytometric determination of apoptosis by annexin V/propidium iodide double staining.

Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde

T H E J O U R N A L O F C E L L B I O L O G Y

Supplementary Materials

At E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in

APA105Hu01 100µg Active Nerve Growth Factor (NGF) Organism Species: Homo sapiens (Human) Instruction manual

SensoLyte Anti-alpha-Synuclein Quantitative ELISA Kit (Human/Mouse/Rat) *Colorimetric*

Nature Medicine doi: /nm.2558

Anti-HB-EGF (Human) mab

Supplemental data. Supplemental Materials and Methods

For Research Use Only. Not for use in diagnostic procedures. Anti-NRF2 mab

Focus Application. Cell Migration. Featured Study: Inhibition of Cell Migration by Gene Silencing. xcelligence System Real-Time Cell Analyzer

Human/Mouse/Rat Phospho-Histone H2AX (S139) Immunoassay

Supporting Information

Stable Isotope Labeling with Amino Acids in Cell Culture. Thermo Scientific Pierce SILAC Protein Quantitation Kits

RNA oligonucleotides and 2 -O-methylated oligonucleotides were synthesized by. 5 AGACACAAACACCAUUGUCACACUCCACAGC; Rand-2 OMe,

An ELISA-based assay using fluorogenic substrates to measure total Cyclooxygenase-2 (COX-2) in whole cells.

SUPPLEMENTARY INFORMATION

For gel-shift assays, 2 ul ivtt synthesized protein (Promega) was incubated at room temperature

used at a final concentration of 5 ng/ml. Rabbit anti-bim and mouse anti-mkp2 antibodies were

CD93 and dystroglycan cooperation in human endothelial cell adhesion and migration

Flag-Rac Vector V12 V12 N17 C40. Vector C40 pakt (T308) Akt1. Myc-DN-PAK1 (N-SP)

Tumor tissues or cells were homogenized and proteins were extracted using

Emanuela Tumini, Sonia Barroso, Carmen Pérez Calero and Andrés Aguilera

1. Goat Anti-Caspase-3 (CPP32) Antibody, R&D systems (cat #AF-605-NA), 0.5ug/ml

SUPPLEMENTARY INFORMATION

Human/Mouse/Rat Phospho-CREB (S133) Immunoassay. An ELISA-based assay using fluorogenic substrates to measure phosphorylated CREB in whole cells.

Supplementary Fig.1 Luton

Supplementary Information: Materials and Methods. GST and GST-p53 were purified according to standard protocol after

Supporting Online Material for

Supplemental Material

HeLa cells stably transfected with an empty pcdna3 vector (HeLa Neo) or with a plasmid

Supplementary information

Supporting Online Material Material and Methods

Protocol for induction of expression and cell lysate production

The Transfection Collection TCF/LEF Transient Pack Wnt / -catenin Signaling Pathway Catalog #: 79273

Single cell imaging of Bruton's Tyrosine Kinase using an irreversible inhibitor

Supplementary material and methods

Supplementary Information

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double mutant mice

Gα 13 Activation Assay Kit

Antibodies used in this study Figure S1. Akt expression in neutrophils from WT and individual Akt isoform knockout mice

Materials Dulbecco s Modified Eagle Medium (DMEM) and fetal calf serum (FCS) were

Checkpoint Kinase Activity Immunoblot Kit

T H E J O U R N A L O F C E L L B I O L O G Y

supplementary information

Supplemental Online Material. The mouse embryonic fibroblast cell line #10 derived from β-arrestin1 -/- -β-arrestin2 -/-

An ELISA-based assay using fluorogenic substrates to measure total inducible Nitric Oxide Synthase (inos) in whole cells.

Supplementary Figure 1 - Characterization of rbag3 binding on macrophages cell surface.

Supplementary Material - Methods

Supplementary Information: Materials and Methods. Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered

Apoptosis assay: Apoptotic cells were identified by Annexin V-Alexa Fluor 488 and Propidium

Lullaby sirna Transfection Reagent - Results

For Research Use Only. Not for use in diagnostic procedures. Anti-NRF2 mab

Supplemental Materials and Methods

EXPERIMENTAL PROCEDURES

Development of Novel Advanced Cell Culture Surfaces that Provide Better Cell Growth and Attachment for Cell-Based Assays

Short hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna

Gα i Activation Assay Kit

Immunoprecipitation Protocol

Supporting Information

NTM486-04, NTM174-04,

Electrophoretic Mobility Shift Assay (EMSA). Nuclear extracts were. oligonucleotide spanning the NF-kB site (5 -GATCC-

Bioimaging of microrna-294 expression-dependent color change. in cells by a dual fluorophore-based molecular beacon

T H E J O U R N A L O F C E L L B I O L O G Y

We performed RT-PCR, cloning, sequencing and qrt-pcr in murine melanoma. cell lines and melanocytic tumors from RET-mice in accordance with the method

Supporting Information

Supplemental methods:

Supplemental Information

SANTA CRUZ BIOTECHNOLOGY, INC.

Supplemental Information: Phosphorylation of CLIP-170 by Both Plk1 and CK2 Is Involved in the Timely Formation of Kinetochore-microtubule Attachments

< Supporting Information >

Cell viability. Cell viability was examined by MTT assay (Sigma-Aldrich).

Histone H3 Methylation Antibody Panel Pack I - Active Genes Base Catalog # C Component Size Shipping Temperature

Four different active promoter genes were chosen, ATXN7L2, PSRC1, CELSR2 and

For Research Use Only. Not for use in diagnostic procedures.

SUPPLEMENTAL MATERIALS SIRTUIN 1 PROMOTES HYPEROXIA-INDUCED LUNG EPITHELIAL DEATH INDEPENDENT OF NRF2 ACTIVATION

DuoSet IC. Human/Mouse Phospho-STAT3 (Y705) Catalog Number DYC4607B-2 Catalog Number DYC4607B-5 Catalog Number DYC4607BE

An ELISA-based assay using fluorogenic substrates to measure total HIF-1 in the context of a whole cell.

Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by

Supplemental Data Supplementary Figure Legends and Scheme Figure S1.

INOS. Colorimetric Cell-Based ELISA Kit. Catalog #: OKAG00807

Supplementary Information (Ha, et. al) Supplementary Figures Supplementary Fig. S1

Supplementary data. sienigma. F-Enigma F-EnigmaSM. a-p53

Gene Forward (5 to 3 ) Reverse (5 to 3 ) Accession # PKA C- TCTGAGGAAATGGGAGAACC CGAGGGTTTTCTTCCTCTCAA NM_011100

DuoSet IC. Human Total p21. Catalog Number DYC Catalog Number DYC Catalog Number DYC1047E

Transcription:

Materials and Methods Antibodies: The following antibodies were used in this study: NuMA - rabbit-anti NuMA (gift from D. A. Compton); Dynein Intermediate Chain - 74.1 (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

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 16-20 hours after transfection. HEK293 cells treated with Colcemid were incubated for 28-36 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

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

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 20 3 5.5 83.2 SK-HEP-1 liver cancer 20 1.5 5.2 85.9 UPCI:SCC078* oral cancer 21 11.3 4.8 52.5 NuMAindependent UPCI:SCC070 oral cancer 28 27 6.6 7.4 JAR placental cancer 10.8 8.6 5.7 6.6 IMR-32 brain cancer 10.6 6.6 1.4 15.9 MES-SA uterine cancer 11.9 12.7 4.8 6.5 4

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 19.9 6.6 28 UPCI:SCC078 oral cancer 22.3 4.8 21 UPCI:SCC103 oral cancer 16.1 5.5 20 JAR placental cancer 14.2 5.7 10.8 I. IMR-32 brain cancer 10.8 1.4 10.6 SK-HEP-1 liver cancer 25.7 5.2 20 MIA-PaCa 2 pancreas cancer 6.0 3.3 12 A549 lung cancer 8.5 3.4 13.8 MES-SA uterine cancer 15.0 4.8 11.9 HCT 116 colon cancer 2.9 4.6 2 5

II. A-431 skin cancer 2.4 9.3 4.3 PC-3 prostate cancer 1.7 11.1 2.2 III. UPCI:SCC114 oral cancer 21.3 89.9 4.7 UP3 oral normal 2.5 96 2.1 IV. GM03349B skin fibroblast 1.0 95.4 4.3 HEK293 embryonic kidney normal 6.1 97.8 9.3 6

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

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

Figure S1

NuMA p150 Glued Dynein IC actin Figure S2

100 Normal extra centrosomes % interphase cells 80 60 40 20 0 untreated Colcemid hmps1 ox untreated HEK293 UPCI:SCC114 Figure S3

A HEK293 B % metaphase cells 100 80 60 40 20 0 untr. hmps1 ox hmps1, NuMA ox bipolar multipolar hmps1, CC1 ox % metaphase cells 100 80 60 40 20 0 untr. hmps1 ox hmps1, NuMA ox Dynein+ Dynein- hmps1, CC1 ox C UPCI:SCC114 D % metaphase cells 100 80 60 40 20 0 untr. bipolar multipolar NuMA ox CC1 ox E % metaphase cells 100 80 60 40 20 0 Dynein+ Dyneinuntr. NuMA ox CC1 ox Figure S4