1. Volume 42, Issue 4, Pages 511-523 (May 2011)
Deubiquitinase USP37 Is Activated by CDK2 to Antagonize APCCDH1 and Promote S Phase Entry 
XiaoDong Huang, Matthew K. Summers, Victoria Pham, Jennie R. Lill, Jinfeng Liu, Gwanghee Lee, Donald S. Kirkpatrick, Peter K. Jackson, Guowei Fang, Vishva M. Dixit 
Molecular Cell 
Volume 42, Issue 4, Pages (May 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 42, 511-523DOI: (10.1016/j.molcel.2011.03.027)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 USP37 Interacts with APC/C through CDH1
(A) Red sequences indicate USP37 peptides identified by mass spectrometry after immunoprecipitation of HA-tagged CDH1 from 293T cells.
(B) Western blot of USP37-FLAG after immunoprecipitation of HA-CDH1 or HA-CDC20 from 293T cells. IP, immunoprecipitation; WCL, whole-cell lysate.
(C and D) Proteins coimmunoprecipitated with USP37-FLAG from 293T cells were identified by mass spectrometry (C) and western blotting (D). Numbers in (C) indicate the frequency of peptide detection in FLAG immunoprecipitates from cells transfected with vector alone, USP6-FLAG, or USP37-FLAG.
(E) Western blot analysis of endogenous proteins in 293T cells immunoprecipitated with a rabbit USP37 polyclonal antibody or a control rabbit IgG.
(F) Western blot analysis of endogenous CDC27 coimmunoprecipitated with endogenous USP37 in 293T cells transfected for 48 hr with control (Ctrl) or CDH1 siRNAs.
(G) USP37-FLAG and HA-CDH1 translated individually in rabbit reticulocyte lysate were mixed together, immunoprecipitated with anti-HA beads, and analyzed by western blotting.
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4. Figure 2 USP37 Regulates the Cell Cycle
(A) HeLa cells were arrested at the G1/S boundary with a double thymidine treatment and then released into fresh medium. Cells were collected every 2 hr.
(B and C) U2OS cells transfected with vector alone or USP37-FLAG for 24 hr were synchronized in prometaphase with nocodazole treatment and then released into fresh medium for the times indicated. In (C), 10 μM bromodeoxyuridine (BrdU) was added to cells during the last 1 hr of culture. Cells incorporating BrdU into their DNA were counted after immunofluorescence staining with an anti-BrdU antibody. At least 200 cells were examined per slide. Error bars, SEM (n = 3 slides per sample).
(D–F) U2OS cells transduced with a lentivirus encoding a USP37 or control (Ctrl) shRNA were synchronized in prometaphase with thymidine and nocodazole, then released into fresh medium. Cells were stained with propidium iodide and their DNA content determined by flow cytometry (E). BrdU incorporation in (F) was determined as in (C). Error bars, SEM (n = 3 slides per sample).
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5. Figure 3 USP37 Is a DUB for Cyclin A
(A) 293T cells were transfected with empty vector, USP37-FLAG, or USP37 C350S-FLAG (USP37DD-FLAG) for 24 hr. Where indicated, cells were treated with 25 μM MG132 for 2 hr prior to harvest.
(B) 293T cells were transfected with four different USP37 siRNAs or a control (Ctrl) siRNA for 48 hr. Numbers indicate the intensity of the cyclin A bands by densitometry. CCNA mRNA encoding cyclin A was determined by TaqMan assay and normalized to GAPDH mRNA. Levels are expressed relative to Ctrl siRNA-transfected cells. Error bars represent SEM of triplicate measurements.
(C) Western blot analysis of endogenous proteins in 293T cells immunoprecipitated with a control rabbit IgG or rabbit polyclonal antibodies recognizing USP37 or cyclin A.
(D) 293T cells were transfected with Myc-cyclin A, HA-ubiquitin (HA-Ubi), USP37-FLAG, or USP37DD-FLAG for 48 hr and incubated with 25 μM MG132 for 1 hr prior to harvest. SDS- and heat-denatured lysates were immunoprecipitated with anti-HA beads.
(E) HeLa cells were transfected with control (Ctrl) siRNA or USP37 siRNA #2 for 48 hr and then synchronized in G1/S with a double thymidine treatment. Cells were then treated with 10 μg/mL cycloheximide (CHX) and harvested at the times indicated. Plotted cyclin A band intensities were quantified by densitometry.
(F) APC/C purified from HeLa cells was combined in vitro with CDH1, E1, UbcH10, ubiquitin, an energy-regenerating system, in vitro-translated [35S]-cyclin A, and either in vitro-translated EMI1 or USP37-FLAG affinity purified from 293T cells. Wheat germ lysate (WGL) served as a negative control. Modification of cyclin A was determined by autoradiography. Ubi-Al, ubiquitin-aldehyde.
(G) Wild-type USP37 and USP37DD were compared as in (F).
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6. Figure 4 USP37 Is an E2F Gene Target
(A–D) U2OS cells were synchronized in prometaphase with nocodazole for 16 hr and then released into fresh medium. USP37, CCNA, E2F1, and EMI1 mRNA levels were normalized to GAPDH mRNA and plotted relative to levels at 0 hr after release. Error bars represent SEM of triplicate measurements.
(E) U2OS cells were transfected with empty vector or E2F1 for 24 hr. USP37 protein was detected by western blotting. USP37 mRNA was quantitated by TaqMan. Error bars represent SEM of triplicate measurements.
(F) 293T cells were transfected with a wild-type or mutant USP37 promoter region (sequences provided in Figure S3) fused to a luciferase reporter gene, plus E2F1 where indicated. Luciferase activity was measured after 24 hr. Error bars represent SEM of triplicate measurements.
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7. Figure 5 USP37 Is Ubiquitinated by APCCDH1 in Late Mitosis
(A) HeLa cells were synchronized in prometaphase with thymidine and nocodazole, then released in fresh medium containing DMSO vehicle or 25 μM MG132 for the times indicated.
(B) 293T cells were transfected with empty vector, FLAG-β-TRCP1, FLAG-β-TRCP2, HA-CDC20, or HA-CDH1 for 24 hr.
(C) 293T cells were transfected with CDH1 siRNA for 48 hr and then cultured for the times indicated in 10 μg/mL cycloheximide (CHX).
(D) USP37 band intensities in (C) were determined by densitometry and plotted as a function of time.
(E) 293T cells were transfected with USP37-FLAG (wild-type or with the indicated degron mutated) alone or together with HA-CDH1.
(F) Western blot analysis of proteins immunoprecipitated from cells in (E) with anti-FLAG antibody.
(G) 293T cells were transfected with USP37-FLAG (wild-type or the KEN-Box 3 mutant) and then cultured in 10 μg/mL cycloheximide (CHX) for the times indicated.
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8. Figure 6 USP37 Deubiquitinates Itself In trans
(A) 293T cells were transfected with USP37DD-FLAG and USP37-Myc as indicated for 24 hr. Where indicated, cells were treated with 25 μM MG132 for 2 hr prior to harvest.
(B) 293T cells were transfected with USP37DD-FLAG, HA-ubiquitin (HA-Ubi), empty vector, and USP37-Myc as indicated for 48 hr. Cells were treated with 25 μM MG132 for 2 hr prior to harvest. SDS- and heat-denatured cell lysates were immunoprecipitated with anti-HA beads.
(C) APC/C purified from HeLa cells was combined in vitro with CDH1, E1, UbcH10, ubiquitin, an energy-regenerating system, and in vitro-translated [35S]-USP37-FLAG. Modification of USP37-FLAG was determined by autoradiography. Ubi-Al, ubiquitin-aldehyde.
(D) HeLa cells were arrested either in G1/S by double thymidine treatment, or in mitosis with thymidine-nocodazole, and then released for 1 hr in the presence of 25 μM MG132. SDS- and heat-denatured cell lysates were immunoprecipitated with a rabbit anti-USP37 polyclonal antibody. Recombinant K11-, K48-, or K63-linked tetra-ubiquitin control lanes confirmed the immunoblot (IB) specificity of the linkage-specific polyubiquitin antibodies.
(E) USP37-FLAG affinity purified from 293T cells was mixed with K11-, K48-, or K63-linked tetra-ubiquitin at 30°C for the times indicated.
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9. Figure 7 CDK2 Phosphorylates and Activates USP37 in G1/S
(A) RPE cells or HeLa cells were transfected with USP37-FLAG or USP9X-FLAG for 24 hr and then arrested in G1/S or prometaphase. Following affinity purification, equivalent amounts of USP37-FLAG or USP9X-FLAG were assayed for cleavage of ubiquitin-AMC. The average DUB activity of USP37 or USP9X at G1/S was arbitrarily set at 100. Error bars represent SEM of triplicate measurements.
(B) HeLa cells were arrested at G1/S or prometaphase. Prometaphase cells were treated with 25 μM MG132 for 1 hr before harvest. Whole-cell lysates (WCL) were incubated with HA-ubiquitin-VS for 1 hr at 30°C, in the absence or presence of 5 mM N-ethylmaleimide (NEM), and then immunoprecipitated with anti-HA beads.
(C) G1/S and prometaphase HeLa cell lysates were resolved in Phos-Tag gels. Where indicated, lysates were treated with calf intestinal phosphatase (CIP) for 1 hr. The upper band corresponds to phosphorylated USP37.
(D) HA-ubiquitin-labeled FLAG-cyclin A and USP37-FLAG were affinity purified from 293T cells. USP37-FLAG was treated with or without CIP for 1 hr. Ubiquitinated cyclin A was then mixed with USP37 for indicated time.
(E) Phosphorylated USP37 residues identified in HeLa cells transfected with USP37-FLAG for 30 hr and then arrested in G1/S (Thy0), prometaphase (Thy-Noco), or released from G1/S arrest for 16 hr (Thy16). Prometaphase cells were treated with 25 μM MG132 for 1 hr before harvest.
(F) The assay was the same as in (D), using indicated USP37 mutants.
(G) HeLa cells were transfected with cyclin A, B, D, or E for 24 hr, and then active endogenous USP37 was labeled with HA-Ubi-VS. USP37 levels were normalized and equal amounts of USP37 were used for labeling.
(H) USP37-FLAG (wild-type, S628A, or S628D) affinity purified from 293T cells was subject to an in vitro kinase assay with cyclin A/CDK2 (upper panel). Coomassie staining of USP37 served as a loading control (lower panel).
Molecular Cell  , DOI: ( /j.molcel )
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