A. Cell cycle synchronized populations were analyzed for HEF1 localization to the centrosome and mitotic apparatus. HEF1 is indicated in green, and visualized with α-HEF1-SB-R1 antibodies previously described 3. γ-tubulin (red) is used to indicate centrosomes (see arrows). DNA (blue) becomes condensed at mitotic entry. Enlarged views of boxed centrosomes are shown in the bottom right; arrows mark centrosomal location. Images are merged confocal sections. 8 μm scale bar applies to top row, 6 μm scale bar applies to all other images. B. FACS analysis demonstrating asynchronous MCF7 cells, and MCF7 cells synchronized in G1, S, and G2/M phases, as used for immunofluorescence analysis. C. Western analysis of HEF1 levels in the indicated phases of cell cycle, with beta-actin as loading control. The HEF1 doublet represents two phosphorylation-induced isoforms of HEF1: 105kDa and 115kDa (hyperphosphorylated) 10. The broad band migrating at ~ 95 kD is a non-specific, crossreacting species detected with the rabbit polyclonal antibody, described in10 (also see Supplemental Figure 1A). D. Cells transfected with plasmids expressing GFP-HEF1_1-363 or GFP-HEF1_1-405, and stained with antibody to gamma-tubulin (red). Gray lines in lower panels outline cell periphery. Boxed centrosomes enlarged in insets: arrows indicate other examples. E. Fragments of HEF1 analyzed as GFP- or FLAG epitope-tagged fusion proteins, amino acids (a.a.) and degree of localization to the centrosome indicated. The degree was estimated by measurement of signal intensity at the centrosome in the same set of experiments (data not shown). HEF1_1-834 is full length HEF1. *, protein poorly expressed.

A. MCF-7 cells with tetracycline-repressed expression (MCF-7 -tTA) of stably integrated HEF1 or GFP in the presence (+) or absence (−) of tetracycline, measured at 24 or 48 hours after medium change. Western analysis with antibody to HEF1 demonstrates induction following tetracycline removal: the beta-actin protein was used as a loading control. B. Western blot analysis of MCF-7 cells infected by retroviruses expressing HA/thioredoxin - tagged peptide fusion proteins (HA-TRX). Levels of HEF1 are stabilized by specific HA-TRX peptides (P1-HEF1, P2-HEF1), but not by non-specific HA-TRX peptides (P1-NS), or by HA-TRX with no peptide inserted (NP). Antibody to HA shows comparable expression of HA-TRX fusions in all lanes. C. Western blot analysis of MCF-7 cells treated with siRNAs to HEF1 (siHEF1 and siHEF1a) or a scrambled (Scr) or GFP (siGFP) oligonucleotide duplex shows efficient and specific HEF1 depletion at 48h and 72 h time points. The blot was stripped and re-probed with β-actin as a loading control. All lanes shown were run on single gel; white lines here and in following figures indicate excision of intervening bands.

A. MCF-7 cells with tet-repressed HEF1 (tTA-HEF1) were uninduced (top row) or induced by tetracycline removal (bottom 3 rows). Cells were treated to visualize DNA (blue) alpha-tubulin (green) and gamma-tubulin (red) for immunofluorescence; representative mitoses are shown. Scale bar for images in this panel is 4 μM. B. Quantitation of multipolar spindles scored in cells with induced (−Tet) or un-induced (+Tet) HEF1 or GFP expression, or treatment with peptides stabilizing endogenous HEF1: specific for HEF1 (H), non-specific (NS), or peptide negative control (NP) HA-TRX fusions. The histogram indicates the percentage of cells with multipolar spindles under different conditions. Red bars indicate conditions with increased HEF1 levels. 3 independent experiments were performed, resulting in the assessment of 150 mitoses in total for each condition. ***, P < 0.001 versus negative control condition. C. tTA-HEF1 cells were first synchronized in double thymidine blocked, then released into medium with tetracycline (blue, HEF1 repressed) or without tetracycline (red, HEF1 induced), and the number of centrosomes scored at 0, 24, or 48 hours. 150 cells were counted in 3 experiments. ***, P < 0.001 difference minus versus plus tetracycline for each time point; difference at 0 hours is not significant. D. tTA-HEF1 cells were presynchronized in thymidine, then placed in medium with hydroxyurea, and with or without tetracycline, and centrosomes scored up to 72 hours. 100 cells were counted in 3 experiments. No significant differences were seen.

A. MCF-7 cells were transfected with either a control scrambled (Scr, top panels) siRNA, or an siRNA specific for HEF1 (siHEF1; center and bottom panels) for 48 hours, then processed for immunofluorescence using markers to gamma-tubulin (red), alpha-tubulin (green), and DNA (blue). Shown, representative mitoses. Scale bar for images in this panel is 5 μm. B. Immunofluorescence analysis of MCF-7 cells with stably expressed GFP-centrin2 (green) 48 hours post-transfection of scrambled (Scr) or HEF1-directed (siHEF1) siRNAs was used to calculate the frequency of split (top panel) versus closely paired (bottom panel, Scr) centrioles (indicated by arrows, with boxed examples shown in insets). The centrioles were considered as split if the distance between them was 2 μm or more 12. Results shown are for depletion with siHEF1; comparable phenotypes are seen with siHEF1a. DNA is shown in blue. Scale bar for images in this panel is 5 μM. C. For quantification, >150 cells were counted from each of at least 3 separate experiments for HEF1-depleted (siHEF1) or control (Scr) siRNA treated cells, and 2 experiments for si p130 and siGFP. All counting was done at a time point after 48h of siRNA treatment. ***, P < 0.001 for siHEF1 versus either Scr or siGFP; */**, P < 0.01 versus Scr and P <0.05 versus siGFP. D. FACS analysis of cells depleted with indicated siRNAs at 48 hours post-transfection.

A. MCF-7 cells were depleted with HEF1-directed siRNA (siHEF1) or a scrambled control (Scr). Mitotic cells stained with antibodies directed at AurA (green), phospho-AurA/T288 (P-AurA, red), and DNA (blue) are shown. Scale bar (5 μm) applies to all panels. B. MCF-7 cells were transfected with scrambled control (Scr) or HEF1-directed (siHEF1) siRNA for 48 hours, then collected and part of the sample elutriated. Non-elutriated (Asyn.), G1/S, or G2-M enriched populations were used for Western blot analysis using the antibodies indicated (left). White vertical lines indicate where intervening (irrelevant) lanes run on the gel have been excised from image. C. MCF-7 cells prepared as in B were used for co-immunoprecipitation. Top, immunoprecipitation with control IgG or Cas antibody (cross-reactive with p130Cas and HEF1; efficient direct precipitation of both proteins), and Western blot using antibodies as indicated to left. Bottom, immunoprecipitation with control IgG or AurA antibody, and Western blot as indicated. For each experiment, co-immunoprecipitation is compared in cells depleted for HEF1, or treated with scrambled siRNA, to confirm specific requirement of HEF1 in co-immunoprecipitation.

A. In vitro translated ³⁵S-labelled AurA was used for pulldowns with GST-fused fragments of HEF1 or GST. Autoradiography shows AurA (top panel) and Coomassie blue (CB) shows GST-fusions (bottom panel). In lane “R”, 20% of a total AurA ³⁵S-labelled reaction mixture is shown: for each pulldown, a complete reaction mixture is used. B. Decreasing quantities (4, 2, or 1 μg) of GST-fused HEF1_1-363 or GST were used in an in vitro kinase reaction with recombinant AurA. Reactions were visualized with antibody to AurA or phospho-AurA/T288, or with CB to show GST-fusions. Phosphorylated histone H3 was visualized by autoradiography. C. AurA was immunoprecipitated from MCF7 cells that were tetracycline repressible for vector (tTA) or HEF1 (tTA-HEF1) expression, + or −tet; and additionally, from cells treated with siRNA to HEF1 (siHEF1) or to scrambled control (Scr). The immunoprecipitated AurA was incubated with recombinant histone H3 with γ-³²P-ATP in an in vitro kinase assay, resolved by SDS-PAGE, and visualized by antibody to AurA, Coomassie blue (CB) staining to detect H3, and autoradiography. D. GST, GST-fusion proteins, or histone H1 or H3 (CB, left) were incubated with AurA and γ-ATP-³²P in vitro. Right, autoradiograph of phosphorylated proteins. E. GST- HEF1_1-363 wild type (wt), or with ala or glu mutations [S296A (A), S296A/S298A (AA), S296E (E), S296E/S298E (EE)] were incubated with AurA and γ-ATP-³²P in vitro. Top, CB shows GST-HEF1 fusions; Bottom, autoradiograph of phosphorylated GST-fusions. F. The GST-fused derivatives of HEF1 described in E were mixed with recombinant AurA and histone H3 (H3) in the presence of γ-ATP-³²P, incubated, and level of histone-H3 phosphorylation determined by autoradiography. Top, CB shows GST-fusions; second row, input histone H3; third row, phospho-histone H3. After incubation, immunoprecipitated AurA (IP:AurA) was probed with antibody to AurA and GST. G. GFP-fused full length HEF1 (wt or with mutations described in E) were transfected into MCF7 cells. After 24 hours, sequential western blot analysis of immunoprecipitated AurA was done using anti-AurA and anti-GFP (top and middle panels). Bottom, anti-GFP shows expression of GFP-fusions in total lysate; bands at ~70–80 kD are degradation products.

A MCF7 cells with integrated GFP-centrin were treated with scrambled control siRNA (Scr), or siRNA to HEF1 (siHEF1), and stained for immunofluorescence with antibodies to HEF1, c-Nap1 or Nek2, as indicated. Scale bars equal 10 μm. B. MCF7 cells were either treated with siRNAs (left panel), or infected with retrovirus expressing full length HEF1 or HEF1 truncations, as for Supplemental Figure 6C. Nek2 kinase was immunoprecipitated, and used for in vitro kinase assays with myelin basic protein (MBP) as a substrate. A comparable level of HEF1 or AurA protein depletion (Supplemental Fig. 5A), and similar levels of overexpression of HEF1 and truncations (not shown) was confirmed with antibodies to GFP and/or HEF1and AurA, in whole cell lysates. C. Antibody to Nek2 or to Cdc2 was used for immunoprecipitation from asynchronous MCF7 cells. Immunoprecipitates were probed with antibodies to HEF1, Nek2, or Cdc2, as indicated; top, middle, and bottom panels.

For histograms *** represents P < 0.001, ** represents P < 0.01, and * represents P < 0.05, in reference to the GFP control. A. Centrosomal splitting in GFP-positive cells transfected with HEF1 truncations. Top, MCF7 cells; bottom, HeLa cells. Bars indicate split centrosomes. Three (MCF7) and two (Hela) independent experiments were performed (n = 100 cells/experiment). B. Cell spreading in GFP-positive cells transfected with HEF1 truncations. Top, MCF7 cells; bottom, HeLa cells. 100 cells were counted in each of 3 experiments. Cell spreading analysis was performed as described in 13, with calculation of area based on pixels within a traced cell perimeter. IF with α-paxillin was used to confirm increased size and formation of focal adhesions in cells plated on laminin and fibronectin. C. Western analysis of expression of different HEF1 truncations expressed as GFP-fused proteins in MCF7 cells. Blot probed with antibody to GFP. Lanes are GFP-fusions to 1, 654-834; 2, 351-363; 3, 1-363; 4, FL; 5, 1-405. D. Mitotic spindle defects induced by HEF1 overexpression in cells plated on different matrices. Tet-repressible MCF-7 derived cells were grown in the presence (+) or absence (−) of tet 48 hours after plating on glass coverslips (−), or coverslips coated with poly-L-lysine (PLL), fibronectin (FN), or laminin (LAM). Bars, multipolar or malformed spindles. 100 mitotic cells were counted in each of 3 independent experiments. E, F. Centrosomal splitting induced by depletion of HEF1 (siHEF1), or overexpression of dominant negative HEF1 (GFP-fused HEF1_1-405), in cells plated on different matrices. For E, MCF7 cells with integrated GFP-centrin2 were transfected with siRNA containing scrambled sequence (Scr) or targeted to HEF1 (siHEF1). For F, MCF7 cells were transfected with GFP-fused HEF1_1-363 or HEF1_1-405, and centrosomes visualized by antibody to gamma-tubulin. Following treatment, cells were plated on glass coverslips (−), or coverslips coated with poly-L-lysine (PLL), fibronectin (FN), or laminin (LAM). Bars, split centrosomes. 100 cells were counted in each of 3 experiments. For D–F, the differences induced by matrix were not statistically significant, except *, P < 0.05 for PLL in D.