Fig 2: USP20 promotes genome stability and suppresses xenograft tumor growth. (A) Depletion of USP20 promotes cell proliferation. A549 cells were transfected with siCTR, si1-USP20 or si2-USP20, transfectants were split 2 days later into a 96-well plate (5000 cells per well) and treated 16 h later with different doses of HU for 72 h. Cell proliferation was determined by the MTT assay. The USP20 knockdown efficiency is shown in Supplementary Figure S10. (B and C) Depletion of USP20 compromised UV-induced intra-S-phase checkpoint. Mock- or USP20-depleted A549 cells described in panel (A) were UV irradiated with a dose of 20 J/m2, pulse-labeled 40 min later with BrdU for 20 min, fixed with 75% ethanol and stained with an anti-BrdU antibody and DAPI (B). BrdU-positive cells in randomly selected fields (more than 100 cells per treatment) were quantified in (C). Three independent experiments were performed. (D–F) USP20 depletion resulted in chromosome aberrations. A549 cells with mocked- or shRNA-mediated USP20 depletion (D) were subjected to a mitotic spread assay. Representative metaphase spreads were shown in (E) and the arrows pointed to the chromosome aberrations. A histogram summarizing percentages of spreads containing aberrant chromosomal structures was shown in (D). More than 80 spreads were randomly selected for analysis. Three independent experiments were performed. (G and H) USP20 suppressed xenograft tumor growth. Note that 5 × 106 mock-depleted (shCTR) or USP20-depleted (sh-USP20) HCT116 cells were injected subcutaneously into the right lower flanks of BALB/c nude mice. The tumor volume was measured starting from day 9 after injection and ending at day 51 at a frequency of twice a week. Tumor volumes were shown as mean ± SEM, and the Student's t-test was performed. Tumor images were shown in (G), while quantification results were shown in (H). *P < 0.05.

Fig 3: USP20 promotes CHK1 activation in response to replication stress. (A and B) Depletion of USP20 delayed HU-induced CHK1 activation. 293T cells were transfected with a control siRNA (siCTR) or a USP20-specific siRNA (si1-USP20), cells were treated with HU 2 days after transfection. Total cell lysates were harvested at different time points after HU treatment and used for immunoblotting with antibodies as indicated. The efficiency of USP20 knockdown was determined by immunoblotting with antibodies against USP20 and β-actin (A). (C) Expression of siRNA-resistant form of USP20 rescued USP20-depletion-induced delay of CHK1 activation in response to HU treatment. 293T cells were transfected with siCTR or si1-USP20. USP20-depleted cells were introduced 48 h after the first transfection with FLAG vector or FLAG-USP20res, the transfectants were treated 2 days later with HU at different time points. Total cell lysates were harvested and used for immunoblotting with antibodies as indicated. (D) Overexpression of the catalytically inactive mutant USP20(C154S) attenuated HU-induced CHK1 activation. 293T cells were transfected with FLAG-USP20 or FLAG-USP20(C154S). Cells were treated 2 days later with HU at different time points. Total cell lysates were harvested and used for immunoblotting with antibodies as indicated.

Fig 4: USP20 promotes CLASPIN stability through deubiquitination. (A and B) USP20 interacted with CLASPIN. Total cell lysates were extracted from 293T cells (A) or 293T cells co-transfected with MYC-USP20 and FLAG-CLASPIN (B), and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. (C) Schematic structure of CLASPIN fragments. (D) The N terminus of CLASPIN (1–330 AAs) mediated the interaction with USP20. Total cell lysates extracted from 293T cells co-transfected with MYC-USP20 and FLAG-CLASPIN or its fragments were subjected to immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with an anti-MYC antibody. (E and F) Inhibition of USP20 expression decreased CLASPIN protein levels and shortened CLASPIN half-life. The siCTR- or si-USP20-transfected 293T cells were treated with CHX at different time points. Total cell lysates were harvested and used for immunoblotting with antibodies as indicated in (E), and quantification of the CLAPSIN/β-ACTIN ratio was plotted in (F). (G) USP20 deubiquitinated K48-linked polyubiquitination of CLASPIN in vivo. 293T cells were co-transfected with the expression constructs as indicated, total cell lysates were harvested 2 days later and subjected to immunoprecipitation followed by immunoblotting with antibodies as indicated. (H) USP20 depletion-induced delay of CHK1 activation in response to HU treatment was reversed by overexpression of CLASPIN. USP20-depleted 293T cells were transfected with FLAG-VEC or wild-type FLAG-CLASPIN and treated 2 days later with HU at different time points. Total cell lysates were extracted and used for immunoblotting with antibodies as indicated.

Fig 5: ART motif in β-arrestin2 (β-arr2) is dispensable for partner protein interactions but required for agonist-induced ubiquitination.A, schematic comparing the location and sequence of ART motif in β-arr2 and yeast arrestin-related trafficking protein 1 (Art1). B, location of the ART motif shown in green traversing across β-arr2 molecule. Structure coordinates from 3P2D were used to generate molecular figure using PyMOL (DeLano, W. L. (2012) The PyMOL Molecular Graphics System v. 1.3r1, Schrödinger, LLC, New York, NY). C, HA-tagged β-arr2 or β-arr2ΔART expressed in HEK-293 cells was immunoprecipitated, and the associations of endogenous USP20 and TRAF6 were determined by immunoblotting. Lysate blot panels show the expression of USP20, TRAF6, and β-arrs in each sample. Blots presented are from one of three independent experiments. Band signals of USP20 normalized to β-arr2 and of TRAF6 similarly normalized from three independent experiments are summarized as means ± SD in the scatter plots with bars in (D) and (E), respectively. ∗p < 0.05, unpaired t test. F, HA-tagged β-arr2 or β-arr2ΔART was isolated by HA-affinity pull down and immunoblotted for ubiquitin (FK2), MDM2, and β-arr2. Lysates were serially probed for HA, MDM2, and GAPDH as loading control. G, ubiquitin signals in (F) were quantitated and normalized to cognate β-arr2 or β-arr2ΔART and summarized as scatter plots with bars from three independent experiments. ∗p< 0.05 versus 0′ and 15′ samples; two-way ANOVA, Holm–Sidak's test. ART, arrestin-related trafficking adaptor; HA, hemagglutinin; HEK-293, human embryonic kidney 293 cell line; MDM2, mouse double minute 2; TRAF6, tumor necrosis factor receptor–associated factor 6; USP20, ubiquitin-specific peptidase 20.