- PDF Free Download

Size: px

Start display at page:

Download ""

Justin Stone
5 years ago
Views:

a Control IgG Intestine c Testis b Thymus 1 3 2 S S 2 1 3 4 4 Figure S1 The wild-type mouse

The mixture was incubated at 4 C for 16 hr or overnight.

CA) using Diaminobenzidine. For counter staining, hematoxylin was used.

villi, which contain stem cells that continuously divide by mitosis.

(b) In thymus, both and proteins are expressed in the immature T stem cells concentrated under

They are also expressed in a small number of round cells both in the inner cortex (2) and

1 a Control IgG Intestine c Testis b Thymus S S Figure S1 The wild-type mouse (C57BL/6J) organs (intestine, thymus and testis) were frozen in liquid nitrogen and sectioned at 5 µm on a cryostat. Immunostaining was carried out using a polyclonal rabbit antibody that was raised against and proteins. The primary antibody was used at 1:5, dilution, except staining of testis at 1:1, dilution. The mixture was incubated at 4 C for 16 hr or overnight. Staining was developed by using the Vectastain Elite ABC kit (Vector Laboratories, Burliname, CA) using Diaminobenzidine. For counter staining, hematoxylin was used. (a) In the small intestine, both and proteins are expressed in the crypts at the base of the villi, which contain stem cells that continuously divide by mitosis. The controls were stained without the first antibodies against and. Bar = µm. (b) In thymus, both and proteins are expressed in the immature T stem cells concentrated under the capsule in the outer cortex (1, 4). They are also expressed in a small number of round cells both in the inner cortex (2) and medulla of thymus (3). Squares are enlarged at the right. Bar = µm. (c) Immunostaining of testis. Squares are enlarged at the bottom. The cells in the outer layer of the seminiferous tubules are spermatogonia, undergoing mitotic division (open arrow heads), whereas sperm heads are observed near the centre of the tubules (S). Spermatocytes between these cells are in meiotic stage (black arrow head). Bar = 5 µm. 1

a 6 h of maturation DNA b 14 h of maturation DNA Control Control Metaphase II Metaphase I - c Figure S2 6 h of maturation 9 7 5 3 1 : Control - (n = 38) (n = 31) (n = 25)

1) Metaphase I - Ana/Telophase I Metaphase II Mis-alignment d 9 7 5 3 1 Oocytes with metaphase II misalignment + + (n = 1) (n = 44) (n =7) (n = 24) Metaphase II

sirna treatment diminished only signals but not signals and vice versa, indicating that and localize to centromeres independently. DNA was stained with TP3. Bar = 1 µm.

Note that only metaphase I/II, but not prometaphase I/II, chromosomes are counted as proper alignment.

2 a 6 h of maturation DNA b 14 h of maturation DNA Control Control Metaphase II Metaphase I - c Figure S2 6 h of maturation : Control - (n = 38) (n = 31) (n = 25) (n = 25) 14 h of maturation / * ** ** Control - (n = 7) (n = 55) (n = 33) (n = 33) (*p <.5, **p <.1) Metaphase I - Ana/Telophase I Metaphase II Mis-alignment d Oocytes with metaphase II misalignment + + (n = 1) (n = 44) (n =7) (n = 24) Metaphase II misalignment Figure S2 Mouse oocytes transfected with, or - sirnas were cultured for 6 h (a) or 14h (b) for maturation, fixed and labeled with anti- and anti- antibodies. sirna treatment diminished only signals but not signals and vice versa, indicating that and localize to centromeres independently. DNA was stained with TP3. Bar = 1 µm. (c) The frequencies of oocytes at various cell cycle stages and metaphase II chromosome misalignment are counted. Note that only metaphase I/II, but not prometaphase I/II, chromosomes are counted as proper alignment. (d) The rate of metaphase II misalignment in - or -depleted oocytes in c were classified by immunostaining into the Sgo signal-positive (+) or negative (-) groups, and the rate of oocytes showing metaphase II misalignment in each group is shown. In this assay, one oocyte of and 2 of were still during meiosis I and therefore excluded. 2

3 ACA DNA control ACA DNA Metaphase I Figure S3 Oocytes with or without depletion of were cultured for 6 h, fixed and stained with anti- antibody and ACA. DNA was counterstained with TP3 iodide. Bar = 1 µm. 3

4 PP2A-C DNA PP2A-C Figure S4 Oocytes cultured for 14 h were fixed and stained by anti-pp2a-c and anti- antibodies. DNA was counter-stained with TP3 iodide. Bar = 1 µm. 4

5 DNA ACA Transient ACA Complete Figure S5 HeLa cells were treated with MG132 and immunostained with anti-, anti- and ACA antibodies, as in Figure 5c. This metaphase cell shows an intermediate separation of centromeres, frequently showing transient redistribution as well as complete redistribution of (enlarged picturesat the right). This suggests that redistribution is the reason, rather thanthe consequence, of centromeric separation. Bar = 1 µm. 5

a control Mad2 c Separated ACA Mad2 Tubulin MG132 MG132

MG132+nocodazole ACA DNA Figure S6 Cells were mock-treated or

thymidine for 24 hr, and released for 12 hr.

(red). DNA was counterstained withhoechst 33342 (blue).

6 a control Mad2 c Separated ACA Mad2 Tubulin MG132 MG132 +nocodazole Mad2 MG132 +nocodazole b Mad2 MG132 MG132+nocodazole MG132+nocodazole ACA DNA Figure S6 Cells were mock-treated or treated with Mad2 sirna, incubated for 6 h, treated with2 mm thymidine for 24 hr, and released for 12 hr. Mitotic cells were harvested byshake-off and treated with the indicated drugs for 5 h. (a) Cells were analyzed bywestern blot with anti-mad2 and anti-tubulin antibodies. (b) Cells were spun ontoglass slides and immunostained with ACA (red). DNA was counterstained withhoechst (blue). Bar = 1 µm. (c) The frequency of separated ACA signals(distance > 1. µm) is shown. Error bars indicate s.e.m. (n = 5 cells, 1 kinetochoreswere scored in each cell). 6

7 Note S1 It is notable that chromosomes in MG132-arrested cells stayed side-by-side even though the prophase pathway may act on the whole chromosome length. This can be explained by the recent finding that prophase pathway is not sufficient to completely remove chromosomal cohesin on the arm region but requires basal activation of separase, which can be blocked by MG132 treatment 1 ). It is also notable that, in contrast to mitotic metaphase arrest, centromeres do not separate at metaphase II arrest in spite of the efficient relocation of shugoshins. In mouse oocytes, however, cohesin Rec8 is not released from chromosome arms during the prolonged metaphase I arrest caused by the inactivation of separase 2 ), implying that the prophase pathway is already absent at the stage of metaphase I and presumably metaphase II as well. This fact can explain why centromeric Rec8 persists throughout metaphase II in spite of the relocation of ; separase must be activated to remove chromosomal Rec8 in either metaphase I or metaphase II stage. References: 1) Nakajima, M., Kumada, K., Hatakeyama, K., Noda, T., Peters, JM. and Hirota, T., The complete removal of cohesin from chromosome arms depends on separase J Cell Sci. doi: /jcs (7) 2) Kudo, N et al., Resolution of chiasmata in oocytes requires separase-mediated proteolysis Cell 126, (6)

Figure S1. HP1α localizes to centromeres in mitosis and interacts with INCENP. (A&B) HeLa

SUPPLEMENTARY FIGURES Figure S1. HP1α localizes to centromeres in mitosis and interacts with INCENP. (A&B) HeLa tet-on cells that stably express HP1α-CFP, HP1β-CFP, or HP1γ-CFP were monitored with livecell