1. The Prolyl Isomerase Pin1 Functions in Mitotic Chromosome Condensation
Yu-Xin Xu, James L. Manley 
Molecular Cell 
Volume 26, Issue 2, Pages (April 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Pin1 Associates Strongly with G2/M Phase Chromatin during the Cell Cycle
HeLa cells were arrested at the G1/S boundary, then released into cell cycle. Every 2 hr, cells were harvested and treated with formaldehyde for ChIP. To obtain pure M phase cells, nocodazole was added to the culture media at 8 hr and cells harvested at 12 and 14 hr (M, lanes 10 and 11). To obtain pure G1 phase cells, nocodazole-treated cells at 14 hr were washed and incubated with normal media for 4 hr (G1, lane 12).
(A) ChIP was performed with anti-Pin1 antibodies, and precipitated DNA was amplified with primers for the β-actin gene at the promoter, coding, and poly(A) regions (top three rows) and cyclin D1 gene in the coding region (bottom).
(B) The same extracts as in (A) were immunoprecipitated with anti-E2F1 antibodies. DNA was amplified with primers for the RPA2 gene at the promoter (top) and poly(A) regions (bottom).
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3. Figure 2
Pin1 Colocalizes with Phosphorylated TopoIIα and CAP-C on Mitotic Chromosomes
(A) Mitotic chromosomes spreads were prepared from HeLa cells arrested with colcemide and stained with anti-Pin1 and MPM2 antibodies.
(B and C) Same preparations as in (A), but stained with anti-TopoIIα and CAP-C antibodies, respectively. The scale bar represents 5 μm. White arrows indicate condensation intermediates, and blue arrows denote mature mitotic chromosomes.
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4. Figure 3 Pin1 Modulates Phosphorylation of Mitotic Phosphoproteins
(A) HeLa tet-on cells expressing flu-tagged Pin1 were cultured in the presence of 1 μg/ml doxycycline and harvested at the times indicated (lanes 2–5). Whole-cell lysates were prepared from the induced cells and from uninduced cells (lane 1). Blots were probed with anti-MPM2, -cdc2, -cdc2 phosphorylated at T14/Y15, and -Pin1 antibodies (from top to bottom, respectively). (Bottom) In vitro histone H1 kinase assay with cdc2/cyclin B immunoprecipitated from the indicated extracts.
(B) Similar to (A), but with cells expressing the R68,69A mutant Pin1.
(C) FACS analyses of wild-type and mutant Pin1-expressing cells without or with doxycycline induction as indicated.
(D) Whole-cell lysates were prepared from HeLa cells transfected with Pin1-specific (C1, C1+C2, lanes 2 and 3, respectively) and control (lane 1) siRNAs, and blots were probed with anti-Pin1 (upper), -MPM2 (middle), and -cdc2 (lower) antibodies. (Bottom) FACS analysis of control- or Pin1-specific siRNA-treated cells.
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5. Figure 4 Pin1 Modulates Phosphorylation of TopoIIα
(A) Whole-cell lysates were prepared from HeLa cells transfected with Pin1-specific (C1, C1+C2, lanes 2 and 3, respectively) or control (lane 1) siRNAs, and blots were probed with a monoclonal antibody specific for Ser1212-phosphorylated TopoIIα (pTopoIIα, top), a polyclonal antibody for TopoIIα (middle), and an antibody for actin (bottom).
(B) Whole-cell lysates were prepared from the stable cell lines analyzed in Figure 3A. Blots were probed with antibodies against phosphorylated TopoIIα, TopoIIα, actin, and Pin1 (from top to bottom, respectively).
(C) Similar to (B), but with cells expressing the R68,69A mutant Pin1.
(D) CoIP of Pin1 with TopoIIα. G1/S phase nuclear (S) and mitotic (M) extracts were prepared from double thymidine-blocked and nocodazole-treated HeLa cells, respectively. Aliquots were immunoprecipitated with anti-Pin1 antibody or rabbit IgG (Mock). The blot was probed with an anti-TopoIIα antibody.
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6. Figure 5 Pin1 Is Necessary for Chromosome Condensation
(A) FACS analysis of HeLa cells at G1/S phase. HeLa cells were arrested at G1/S phase (left), then released into the cell cycle for 6 hr (right).
(B) Depletion of Pin1 from mitotic extract (ME). MEs were prepared from HeLa cells arrested with nocodazole. Aliquots were immunoprecipitated with anti-Pin1 antibody or rabbit IgG (Mock). Pellets (20%) and supernatants (5%) were fractionated by SDS PAGE. Blots were probed with anti-Pin1 (upper) and -cdc2 (lower) antibodies.
(C) HeLa cells arrested at G1/S phase were released for 6 hr, and cells on coverslips were treated with 0.5% Triton X-100/PBS. Nuclei were then incubated with buffer (upper), ME (middle), or mock-depleted ME (lower) for 3 hr and then fixed and stained with PI. Arrowheads indicate nuclei shown at higher magnification to the left.
(D) Similar to (C), but with Pin1-depleted ME (D-ME) for 1, 2, and 3 hr (from top to bottom, respectively).
(E) Similar to (C), but with D-ME plus wild-type Pin1 for 1, 2, and 3 hr (from top to bottom, respectively).
(F) Similar to (C), but with D-ME plus R68,69A mutant Pin1 for 3 hr.
(G) Quantitation of chromosome condensation. Nuclei from experiments in (C)–(F) were analyzed as described in Figure S2. Bars represent the percentage of fluorescence intensity less than a threshold value across intact nuclei. Averages and standard deviations were calculated from at least five randomly selected nuclei.
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7. Figure 6 Pin1 and Cdc2/Cyclin B Induce Chromosome Condensation
(A) HeLa nuclei were prepared as in Figure 5C. Nuclei were incubated with purified cdc2/cyclin B for 1, 2, and 3 hr (from top to bottom, respectively), fixed, and stained with PI. Arrowheads indicate nuclei shown at higher magnification to the left.
(B) Similar to (A), but with cdc2/cyclin B plus wild-type Pin1 for 1, 2, and 3 hr.
(C) Similar to (A), but with cdc2/cyclin B plus the R68,69A Pin1 mutant for 3 hr.
(D) Quantitation of chromosome condensation. Analysis as in Figure 5G, but with nuclei from (A)–(C).
(E) Cdc2 immunoprecipitated from reaction mixtures with S phase nuclei without or with Pin1 (lanes 2 and 3, respectively) as in (B) were analyzed by western blotting. The blot was first probed with antibody for cdc2 phosphorylated at T14/Y15 (top) and then with antibody for cdc2 (bottom). Lanes 1 and 4 are untreated S phase and MEs, respectively.
(F) In vitro histone H1 kinase assays with cdc2/cyclin B immunoprecipitated from S (lane 1) or M (lane 2) phase extracts and from reaction mixtures with S phase nuclei plus flu-cdc2/cyclin B without (lane 3) or with (lane 4) Pin1.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Mechanism of Pin1 Function in Chromosome Condensation
(A) S phase nuclei were treated with buffer or the indicated extract as in Figure 5 and stained with antibodies for TopoIIα (left) or histone H3 phosphorylated at Ser10 (right) or with PI as indicated.
(B) TopoIIα (200 ng) was incubated with buffer (lane 1), cdc2/cyclin B (∼20 ng, lane 2), or cdc2/cyclin B plus wild-type or R68,69A mutant Pin1 (100, 200, and 400 ng, lanes 3–5, and lanes 6–8, respectively). The blot was probed with an antibody specific for Ser1212-phosphorylated TopoIIα.
(C) TopoIIα (80 ng) was preincubated with buffer (lane 2), cdc2/cyclin B (∼20 ng, lane 3), or cdc2/cyclin B plus wild-type or R68,69A mutant Pin1 (200, 400, and 800 ng, lanes 4–6 and lanes 7–9, respectively). 32P-end-labeled DNA (4 ng) was added and reaction mixtures incubated at RT for 10 min and separated on 1% low melting point agarose gels. Lane 1, DNA incubated in buffer alone. Lane 10, DNA plus 800 ng Pin1 and cdc2/cyclin B. Lanes 11 and 12, DNA plus TopoIIα and 800 ng Pin1, but without or with cdc2/cyclin B, respectively. Lanes 14–16, reaction mixtures as in lane 6 were incubated at RT for 10 min with 50 ng anti-Pin1, anti-TopoIIα, or IgG antibodies. Upper and lower arrows indicate positions of supershifted and unshifted complexes, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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