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

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1 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 Ricke et al., Figure S1. Kinetochore localization of mitotic regulators in wild-type and Bub1 kinase deficient primary MEFs. (A) Analysis of mitotic timing in Bub1 +/+ and Bub1 KD/KD cells with proper chromosome segregation and in Bub1 KD/KD cells with misaligned chromosomes. P, prophase; PM, prometaphase, M, metaphase; A, anaphase. (B) Analysis of mitotic duration in BubR1 +/+ and BubR1 H/H MEFs after colcemid addition. The time at which 50% of the cells exited mitosis was compared. (C) IF of unperturbed MEFs immunostained as indicated. DNA was visualized with Hoechst (blue). (D) IF of chromosome spreads from taxol (left)- or nocodazole (right)-treated MEFs. Chromosomes were stained for Bub1 (green), centromeres (cyan), and DNA (blue). Error bars indicate SEM. KD, kinase dead. Bars, 10 µm. S1

2 Figure S2. Bub1 kinase activity is required for Sgo1 and Aurora B inner centromeric accumulation. (A) Representative images of IF of unperturbed intact cells stained for Sgo1 (green) and DNA (blue). (B) Quantification of the Sgo1 signal from cells described in A using ImageJ from three independent cell lines per genotype. (C) Same as A except with Sgo2. (D) Same as B except with Sgo2. (E, top) Representative images of IF of chromosome spreads from colcemid-blocked MEFs. Chromosomes were stained for Aurora B (green), centromeres (cyan), and DNA (blue). (bottom) Quantification of the number of cells with the indicated Aurora B localization. The mean of three independent cell lines is shown per genotype. Statistics are with comparison with wild type. (F) Same as E except with detergent pretreatment. (G) Representative images of IF of chromosome spreads from taxol-blocked MEFs stained for Aurora B (green), centromeres (cyan), and DNA (blue). (H) Same as G but with detergent pretreatment. Error bars indicate SEM. a.u., arbitrary unit; KD, kinase dead; cent, centromere. *, P < 0.5; **, P < 0.1. Bars, 10 µm. S2

3 Figure S3. Bub1 kinase activity is required for Aurora B inner centromeric accumulation. (A) Representative images of IF from intact cells stained for Aurora B (green), centromeres (red), and DNA (blue). (B) Quantification of the Aurora B signal described in A using ImageJ. The mean of three independent cell lines per genotype is shown. (C) Western blot analysis of lysates from MEFs probed for Bub1, BubR1, Aurora B, INCENP, Sgo1, actin, and ph3 S10. (D) Western blot analysis of lysates from MEFs probed for Bub1, ph3 T3, and ph3 S10. (E) Haspin overexpression does not rescue Aurora B localization in the absence of Bub1 kinase activity. The number of cells with the indicated Aurora B localization was quantified after transduction and selection with lentivirus encoding Haspin (VSV-Haspin) or empty vector (EV). The mean of three independent cell lines is shown per genotype. (F) IF on intact cells stained for survivin (green), centromeres (red), and DNA (blue). (G) Bub1 +/+ and Bub1 KD/KD cells were infected with and selected for lentivirus encoding HA-survivin. The number of cells with the indicated HA-survivin localization in Bub1 +/+ (+/+) or Bub1 KD/KD (KD/KD) cells that were costained for centromeres and HA-survivin. The mean of three independent cell lines is shown per group. Statistics indicate comparison with wild type. (H) Representative images of IF of chromosome spreads from colcemid-blocked MEFs that were generated as described in G. Chromosomes were stained for Aurora B (green), HA-survivin (red), and DNA (blue). (I) HeLa cells were treated for 6 h with nocodazole alone ( ) or nocodazole (Noc.) with 1 µm or 2 µm ZM. Western blot analysis for ph3 S10 as a marker for Aurora B activity and actin as a loading control. (J) Two independent wild-type primary MEFs were treated for 3 h with nocodazole alone ( ) or nocodazole with 1 µm or 2 µm ZM. Western blot analysis for ph3 S10 as a marker for Aurora B activity and actin as a loading control. Error bars indicate SEM. a.u., arbitrary unit; KD, kinase dead; PM, prometaphase. *, P < 0.5. Bars: (yellow) 10 µm; (red) 1 µm. S3

4 Figure S4. Generation of a conditional Bub1 allele. (A) Bub1 gene targeting strategy for Bub1 floxed allele. Relevant portion of the Bub1 locus (+), the targeting vector, the targeted allele (Bub1 FNEO ), the targeted allele after Cre recombination (Bub1 F ), BamHI (B) restriction sites, loxp sites (red triangles), Flp recombination target sites (blue triangles), and the Southern probe are indicated. NEO, neomycin; Hsv-tk, herpes simplex thymidine kinase. (B) Southern blot analysis of targeted ES clones digested with BamHI. FNEO, loxp flanked neomycin cassette; X, digested. (C) IF of MEFs from Bub1 F/F + HA-Bub1 E252K (HA- Bub1 E252K ) or Bub1 / + HA-Bub1 immunostained for centromeres (cyan), HA (red), or Bub1 (green). DNA was visualized with Hoechst (blue). (D) Western blot of lysates from asynchronous MEFs probed for ph3 S10, HA, actin, and Bub1. (E) Immunostaining of monastrol-treated Bub1 F/F MEFs 4 d after infection with empty vector (EV) or ptsin-cre (Cre) for ph2a T121 (green) and centromeres (cyan). DNA was visualized with Hoechst (blue). (F) Mitotic extracts prepared from MEFs of the indicated genotype. Cells were subjected to immunoprecipitation with anti-ha antibodies and analyzed by Western blotting as indicated. (G) Western blot of lysates from nocodazole-blocked MEFs probed for ph2a T121, ph3 S10, HA, and Bub1. The asterisk indicates that the epitope used to generate the polyclonal Bub1 antibody that overlaps with the deletion in Bub1- Mad3. Note that lane 3 was from the same films as lanes 1 and 2, but the intervening samples were spliced out. (H) Representative images of IF of chromosome spreads from colcemid-blocked MEFs pretreated with detergent stained for Aurora B (green), centromeres (cyan), and DNA (blue). (I) HA-Bub1 was immunoprecipitated from the indicated taxol-blocked immortalized cell lines and incubated with histone H2A in the presence of -[ 32 P]ATP. IP, immunoprecipitation; WB, Western blot. Bars, 10 µm. S4

5 Figure S5. Bub1 kinase deficient male mice are subfertile. (A) The number of litters and pups produced from Bub1 KD/KD males housed with wild-type females for 90 d. Statistics indicate comparison with wild type. Lines mark the means. (B) Same as A except that Bub1 KD/KD females were housed with wildtype males. (C) Testes and spleen weight in 8-wk-old males. Error bars indicate SEM. Statistics are in comparison with wild type. (D) Sperm counts from 8-wk-old males (n = 3). Error bars indicate SEM. Statistics indicate comparison with wild type. (E) Hematoxylin and eosin stained testes sections. Bar, 50 µm. (F) Percentage of aneuploidy in primary and secondary spermatocytes. Statistics indicate comparison with wild type. (G) Representative images of primary spermatocytes from wild type and Bub1 KD/KD mice. Numbers indicate number of chromosome pairs. Bar, 10 µm. KD, kinase dead. *, P < 0.5; **, P < 0.1. S5