T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Posch et al., http://www.jcb.org/cgi/content/full/jcb.200912046/dc1 Figure S1. Biochemical characterization of the interaction between Sds22-GFP and PP1 isoforms and the localization of Sds22-GFP on kinetochores detected with anti-sds22. (A) Physical interaction of GFP-Sds22 and PP1 in cell lysates. Lysates of logarithmically growing HeLa cells transiently transfected with Sds22-EGFP or EGFP were immunoprecipitated with anti-gfp antibody cross-linked to protein G Sepharose. Immunoprecipitates (IP), total lysates and 10% of input (inpt), and depleted (depl) extract loaded for control were probed with anti-gfp or anti-pp1. A nonspecific anti-pp1 band is marked by asterisks. (B) Proteomic analysis of GFP-Sds22 binding proteins immunoprecipitated (GFP-Trap) lysates from mitotically synchronized (10 h after release from double-thymidine block) cell lines stably expressing EGFP and EGFP-Sds22 (clone D103) were analyzed by liquid chromatography mass spectrometry. PP1 isotype specific peptides identified are indicated on the right. (C) Anti-Sds22 stains Sds22-GFP at kinetochores. D103 cells expressing GFP-Sds22 were fixed in formaldehyde and stained with Sds22 (red)-, GFP (green)-, and ACA (blue)-specific antibodies in immunofluorescence. Boxed region is shown at higher magnification views below. Line profile shows relative intensities across the kinetochore pair. Bars: (top) 5 µm; (bottom) 1 µm. Sds22 in mitosis Posch et al. S1
Figure S2. Dose-dependent inhibition of aurora B T232 phosphorylation by ZM. 48 h after transfection with control (con) or Sds22-specific RNAi, cells were incubated in medium containing monastrol for 4 h. Monastrol was washed out with fresh medium and, where indicated, supplemented with the aurora B specific inhibitor ZM at the indicated concentrations. After 45 min, cells were fixed and processed for immunofluorescence with anti phospho-t232 aurora B and ACA antibodies. Overlay shows anti phospho-t232 aurora B (red), ACA (blue), and tubulin (green). Bars, 10 µm. S2
Figure S3. Unaligned pairs 45 min after washout of monastrol. (A) HeLa cells were fixed and immunostained 60 h after transfection with either control or Sds22-specific RNAi and 45 min after monastrol washout. Overlay shows phospho-t232 aurora B (green), ACA (red), and tubulin (white). (left) White boxes show regions displayed in higher magnification on the right. (B) Comparison of aurora B localization by conventional and 3DSIM imaging. Identical cells were imaged either on a DeltaVision Core microscope (DV) or a DeltaVision OMX system in conventional (OMX conv) and 3DSIM (OMX SI) modes. Maximum projections of 0.2 µm sections (DV and OMX conv) or reconstructed 0.125 µm sections (OMX SI) are shown. Overlay shows aurora B (green) and ACA (red). Bars: (A, left) 10 µm; (A, right) 1 µm; (B) 5 µm. Sds22 in mitosis Posch et al. S3
Figure S4. CENP-A phosphorylation is reduced in cells depleted of PP1 isoforms. Intensities of phospho CENP-A in HeLa cells fixed 48 h after transfection with control and PP1 isoform specific RNAi duplexes. Box plots and statistical analysis are as in Fig. 3 (E and F). S4
Figure S5. Effects of Sds22 depletion on aurora B substrates and interkinetochore distances. (A) Ser55-Hec1 phosphorylation in prometaphase and metaphase cells in control (con) and Sds22-depleted cells. Overlay images show ACA (blue), microtubules (green), and anti-phospho Ser55-Hec1 (red). (B) Ser92 MCAK phosphorylation (Andrews et al., 2004) in prophase, prometaphase, and metaphase cells in control and Sds22-depleted cells. Overlay images show ACA (blue), microtubules (green), and anti phospho-mcak (red). (C) Ser44-Hec1 phosphorylation in prometaphase and metaphase cells in control and Sds22-depleted cells. Overlay images show ACA (blue), microtubules (green), and anti-phospho Ser44-Hec1 (red). (D) Interkinetochore distances per cell (mean ± SEM) in a HeLa cell line stably expressing CENP-A GFP. Time-lapse images of late prometaphase or metaphase were analyzed as previously described (Jaqaman et al., 2010). Aligned sister kinetochores were identified, tracked, and assigned to sister pairs, and interkinetochore distances were evaluated using a completely automatic data analysis pipeline. Data from three independent experiments are shown. Total number of cells: control sirna, 32; Sds22 RNAi, 63. Box plots and statistical analysis are as in Fig. 3 (E and F). Bars, 10 µm. Sds22 in mitosis Posch et al. S5
Video 1. Control RNAi. Maximum intensity projections from 3D time-lapse images of GFP CENP-A expressing HeLa cells treated with control RNAi. Corresponds to Fig. 1 D (i). The video is shown at 32 frames per second. Video 2. Sds22 RNAi. Maximum intensity projections from 3D time-lapse images of GFP CENP-A expressing HeLa cells treated with Sds22 RNAi dividing with unaligned chromosomes. Corresponds to Fig. 1 D (ii). The video is shown at 32 frames per second. Video 3. Sds22 RNAi dividing with delay at the midzone. Corresponds to Fig. 1 D (iii). The video is shown at 32 frames per second. References Andrews, P.D., Y. Ovechkina, N. Morrice, M. Wagenbach, K. Duncan, L. Wordeman, and J.R. Swedlow. 2004. Aurora B regulates MCAK at the mitotic centromere. Dev. Cell. 6:253 268. doi:10.1016/s1534-5807(04)00025-5 Jaqaman, K., E.M. King, A.C. Amaro, J.R. Winter, J.F. Dorn, H.L. Elliott, N. McHedlishvili, S.E. McClelland, I.M. Porter, M. Posch, et al. 2010. Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases. J. Cell Biol. 188:665 679. doi:10.1083/jcb.200909005 S6