1. Destruction of Claspin by SCFβTrCP Restrains Chk1 Activation and Facilitates Recovery from Genotoxic Stress 
Niels Mailand, Simon Bekker-Jensen, Jiri Bartek, Jiri Lukas 
Molecular Cell 
Volume 23, Issue 3, Pages (August 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Claspin Is Degraded upon Entry into Mitosis
(A and B) Exponentially growing U-2-OS cells and primary BJ fibroblasts were fixed and immunostained with the indicated antibodies. The nuclear DNA was visualized by counterstaining with the ToPro3 DNA intercalating dye. In (B), cells were grown in the presence of BrdU for the final 1 hr before fixation. Scale bar, 10 μm.
(C) U-2-OS cells were grown exponentially (Exp) or synchronized in mitosis by incubation in the presence of nocodazole for 12 hr. After release from the mitotic block, cells were harvested at various times and probed for the abundance of indicated proteins by immunoblotting. MCM6 serves as a loading control.
(D) Asynchronous (Exp) or nocodazole-arrested (M) U-2-OS cells were fractionated to separate soluble (Sol) or chromatin-enriched (Chrom) proteins, and the resulting extracts were analyzed by immunoblotting with the indicated antibodies. Orc2 was used as a loading control.
(E) U-2-OS/shClaspin cells were kept repressed or induced to express Claspin-specific shRNA by addition of doxycycline (Dox) for 48 hr and harvested, and total cell extracts were probed for the indicated proteins by immunoblotting.
(F) U-2-OS/shClaspin cells treated as in (E) were grown on coverslips, fixed, and analyzed for the indicated proteins in G2 phase, prophase (Pro), and metaphase (Meta) by immunostaining. Scale bar, 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The Early Mitotic Degradation of Claspin Is Mediated by SCFβTrCP
(A) U-2-OS cells were transfected with the indicated constructs for 24 hr and then kept unsynchronized (Exp) or synchronized in mitosis by nocodazole treatment (M). The levels of ectopic proteins were monitored by immunoblotting of total cell extracts.
(B) U-2-OS cells were transfected with FLAG-tagged full-length Claspin (FL) or a deletion mutant lacking the first 100 amino acids (ΔN) and synchronized as in (A). Total cell extracts were probed for the indicated proteins by immunoblotting.
(C) Schematic representation of the conserved βTrCP recognition motif in vertebrate Claspin orthologs.
(D) U-2-OS cells were transfected with FLAG-tagged βTrCP expression plasmid as indicated. Subsequently, FLAG-βTrCP1 was immunoprecipitated with FLAG-resin and analyzed for its ability to interact with Claspin by immunoblotting. Abbreviation: WCE, whole-cell extract.
(E) In vitro-translated (IVT) [35S] labeled Claspin (residues 1–380) was phosphorylated in extracts from exponentially growing or mitotic U-2-OS cells and incubated with GST-βTrCP1 bound GSH beads. βTrCP bound Claspin was resolved by SDS-PAGE and visualized by autoradiography.
(F) In vitro-translated [35S] labeled Claspin(1–380) was phosphorylated in mitotic extracts and treated for 30 min with λ-phosphatase where indicated. The binding of labeled Claspin to GST-βTrCP immobilized on GSH beads was analyzed as in (E).
(G) U-2-OS cells were transfected with control or βTrCP1/2 siRNA for 24 hr and then kept asynchronous or synchronized in mitosis. Cell lysates were analyzed by immunoblotting with the indicated antibodies.
(H) U-2-OS cells grown on coverslips were transfected as in (G) for 48 hr, fixed, and immunostained with the indicated antibodies and counterstained with ToPro3. Scale bar, 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Mitotic Degradation of Claspin Depends on a Conserved βTrCP Degron
(A) Schematic depiction of the βTrCP phosphodegron mutant Claspin 2A protein.
(B) U-2-OS cell lines conditionally expressing wild-type (wt) or mutant (2A) Claspin were induced to express the transgenes for 24 hr and grown exponentially or synchronized in mitosis. Cell lysates were probed for the indicated proteins by immunoblotting.
(C) U-2-OS cells were transfected with the indicated constructs for 24 hr and synchronized as in (B). Protein expression was monitored by immunoblotting.
(D) In vitro ubiquitylation reactions were carried out with extracts from unsynchronized or mitotic U-2-OS cells supplemented with βTrCP and in vitro-translated [35S] labeled Claspin(1–380). Ubiquitylated Claspin was visualized by autoradiography.
(E) U-2-OS cells were transfected with control or βTrCP1/2 siRNA for 24 hr and then synchronized in mitosis. Extracts from these cells were used in in vitro ubiquitylation assays as in (D). Note that the ubiquitylation reaction was carried out without addition of recombinant βTrCP; consequently, the formation of long polyubiquitylated chains (Ubn) was less prominent. Nevertheless, the inefficient Claspin ubiquitylation in the βTrCP-depleted extract is clearly visible as the lack of conversion of unmodified Claspin (marked by asterisk) to higher molecular weight species.
(F) In vitro-translated [35S] labeled wt or mutant N-terminal fragments of Claspin were phosphorylated in mitotic extracts from U-2-OS cells, and their binding to βTrCP was analyzed as in Figure 2F.
(G) U-2-OS/FLAG-Claspin cell lines were induced to express the transgenes for 24 hr and kept unsynchronzied or arrested in mitosis. Cycloheximide (CHX) was added to inhibit protein synthesis, and cells were harvested at the indicated times and analyzed by immunoblotting of total extracts.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
The Polo-like Kinase Plk1 Is Required for the Mitotic Degradation of Claspin
(A) Human 293T cells were transfected with the indicated constructs for 24 hr. Subsequently, Myc-Plk1 was immunoprecipitated with Myc-resin and analyzed for its ability to interact with FLAG-Claspin by immunoblotting.
(B) U-2-OS cells were transfected with indicated siRNAs for 24 hr and then kept nonsynchronized or synchronized in mitosis. Levels of indicated proteins were monitored by immunoblotting.
(C) U-2-OS cells grown on coverslips were transfected as in (B) for 48 hr, fixed, and immunostained with the indicated antibodies and counterstained with ToPro3. Cells in early prophase are indicated with arrows. Scale bar, 10 μm.
(D) In vitro ubiquitylation reactions were carried out as in Figure 3D, using mitotic extracts from U-2-OS cells transfected with control or Plk1 siRNAs.
(E) IVT [35S] labeled Claspin(1–380) was phosphorylated in mitotic extracts prepared from cells transfected with control or Plk1 siRNAs, and the binding between Claspin and GST–βTrCP1 was analyzed as in Figure 2E.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Inability to Degrade Claspin Delays Checkpoint Recovery after DNA Damage and Replication Stress
(A) Exponential U-2-OS cells and derivative lines induced to express FLAG-Claspin wt and 2A for 24 hr were incubated in the presence of nocodazole. The cells were harvested at the indicated times, and mitotic accumulation was determined by immunodetection of phospho-histone H3 using dual-parameter flow cytometry. Results show the mean of two independent experiments.
(B) U-2-OS cells or FLAG-Claspin clones induced to express the transgenes for 24 hr were presynchronized in S phase by incubation in the presence of 3 mM thymidine for 24 hr. Six hours after release from the S phase arrest, cells were pulse treated with 0.25 μM adriamycin for 1 hr, washed extensively, and incubated in fresh medium containing nocodazole. Mitotic entry in cells fixed at the indicated times was monitored as in (A). Results show the mean of two independent experiments.
(C) Extracts from cells treated as in (B) were analyzed by immunoblotting for the indicated proteins.
(D) U-2-OS cells or FLAG-Claspin derivative clones induced to express the transgenes for 24 hr were treated with 2 mM hydroxyurea (HU) for 24 hr. After extensive washing, cells were incubated in fresh medium containing nocodazole, and the proportion of mitotic cells at the indicated times were measured as in (A). Results show the mean of two independent experiments.
(E) Extracts from cells treated as in (D) were analyzed by immunoblotting for the indicated proteins.
Error bars in (A), (B), and (D) indicate twice the standard deviation at the respective time points.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Expression of Stable Claspin Supports Unscheduled Chk1 Phosphorylation
(A) U-2-OS cells were kept unsynchronized or trapped in prometaphase by incubating in the presence of nocodazole for 12 hr. Subsequently, cells were treated with 0.5 μM adriamycin (ADR) for 2 hr, and DNA damage-dependent phosphorylation of Chk1 was analyzed by immunoblotting, using a phosphospecific antibody to Ser317 (Chk1-pS317).
(B) U-2-OS/FLAG-Claspin lines were induced to express the transgenes for 24 hr and synchronized and analyzed as in (A).
(C) U-2-OS/FLAG-Claspin 2A cells were kept repressed or induced to express the transgene for 24 hr, synchronized in mitosis, and treated with 0.5 μM adriamycin for 2 hr. Total cell extracts were analyzed by immunoblotting.
(D) U-2-OS cells were transfected with control or βTrCP1/2 siRNA for 24 hr and synchronized in mitosis by nocodazole shake off. Cells were subsequently treated with adriamycin and analyzed as in (A).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7
Model of βTrCP-Mediated Regulation of Cell Cycle Checkpoint Response
(A) Initiation and maintenance of cell cycle checkpoints relies on βTrCP-mediated degradation of Cdc25A.
(B) Checkpoint recovery and resumption of cell cycle progression is associated with a switch of βTrCP preference to Claspin and Wee1. See text for detailed description of the model.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions