HeLa (cat. no. CRM-CCL2), MCF-7 (cat. no. HTB-22), and 293T (cat. no. CRL-3216; all from ATCC) cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic reagent (containing penicillin, streptomycin, and amphotericin B; cat. no. 15240-062; Gibco: Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ incubator. A549 (cat. no. CRM-CL-185; ATCC) cells were incubated in Roswell Park Memorial Institute 1640 (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.) medium containing 10% FBS, 1% antibiotic-antimycotic reagent (as previously aforementioned) at 37°C in a 5% CO₂ incubator. Transfection was performed using 150 mM NaCl and 10 mM linear polyethyleneimine (PEI; Polysciences, Inc.) and harvested after 48 h.

PP2A gene was subcloned into the pCS4-Flag vector and pcDNA3.1-Myc vector. USP7 and USP7 (C223S, a catalytically inactive mutant) genes were subcloned into the pcDNA3.1-Myc vector (12) or pCS4-Flag vector. USP7 gene was subcloned into the pGEX-4T-1 vector for GST pull-down assay (GST-USP7). The HA-tagged ubiquitin (K48 and K63) was produced using site-directed point mutagenesis (26). HA-K48 is an HA-tagged ubiquitin in which other lysines (K6, K11, K27, K29, K33, and K63) are mutated to an arginine except for K48. While HA-K63 is an HA-tagged ubiquitin in which other lysines (K6, K11, K27, K29, K33, and K48) are substituted with an arginine except for K63. The purpose of HA-K48 and HA-63 is to specifically identify an HA-K48-linked- or HA-K63-linked ubiquitin chain, respectively. Anti-β-actin (cat. no. sc-4778; Santa Cruz Biotechnology, Inc.), anti-HA (cat. no. 12CA5; product code 11583816001; Roche Diagnostics), anti-Flag (cat. no. M185-3L; MBL International Corporation), anti-Myc (cat. no. sc-40; Santa Cruz Biotechnology, Inc.), anti-USP7 (cat. no. CSB-PA849973LA01HU; Cusabio Technology LLC), anti-USP7 (cat. no. sc-137008; Santa Cruz Biotechnology, Inc.), and anti-PP2A (cat. no. sc-6110; Santa Cruz Biotechnology, Inc.) antibodies were used.

Harvested cells were lysed using a lysis buffer (50 mM Tris-HCL, 300 mM NaCl, 1 mM EDTA, 10% Glycerol, 1% Triton X-100) and were incubated for 20 min on ice. Cell lysates were centrifuged at 16,200 × g at 4°C for 20 min, and the supernatant was boiled with 2X SDS buffer at 100°C. The protein amounts (30 µg) were determined by Bradford assay using Bio-Rad protein assay dye reagent (cat. no. 5000006; Bio-Rad Laboratories, Inc.). The samples were run in an 8% SDS-page gel and transferred onto microporous polyvinylidene fluoride membranes (cat. no. IPVH00010; EMD Millipore; Merck KGaA). The membranes were then blocked with 5% skim milk in TBS buffer, including 0.05% Tween-20 for 30 min at room temperature, and incubated at 4°C overnight with a primary antibody [β-actin (1:3,000), HA (1:1,000), Flag (1:5,000), Myc (1:100), USP7 (1:500; cat. no. sc-137008; Santa Cruz Biotechnology, Inc.), and PP2A (1:5,000)] in 2% skim milk with TBS buffer, including 0.05% Tween-20. The probed membranes were incubated in 1% skim milk with TBS buffer including 0.05% Tween-20 with KPL peroxidase-labeled antibody to mouse IgG (H+L) (cat. no. 074-1806; 1:30,000; LGC SeraCare) and mouse anti-goat IgG-HRP (cat. no. sc-2354; 1:30,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The proteins were detected using an ECL reagent solution (Young In Frontier). For immunoprecipitation, 2 mg of cell lysates was incubated with antibodies [Flag, Myc, USP7, and PP2A (1 µg per 500 µg of total proteins)] at 4°C on a rotator overnight. A resuspended volume (30 µl) of Protein A/G PLUS-agarose beads (cat. no. sc-2003; Santa Cruz Biotechnology, Inc.) was then added to the cell lysates and incubated at 4°C on a rotator for 2 h. The samples were then washed with washing buffer [50 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, a protease inhibitor cocktail (PIC; cat. no. 11697498001; Roche Diagnostics), and phenylmethanesulfonyl fluoride (PMSF; cat. no. P7626; Sigma-Aldrich; Merck KGaA)]. Subsequently, the samples were centrifuged at 3,200 × g at 4°C for 5 min, and then boiled in 35 µl of 2X SDS buffer at 100°C for 7 min. The supernatant (25 µl) was then loaded onto 8% SDS-PAGE gel. The remaining steps were the same as those described for the western blotting.

For ubiquitination assay, Flag-PP2A and HA-ubiquitin (HA-Ub) (K48 or K63) were transfected into 293T cells. The HA-tagged lysine mutants have been generated by substituting each lysine site with an arginine except one corresponding lysine site. After 48 h, cells were harvested and lysed using a lysis buffer (50 mM Tris-HCL, 300 mM NaCl, 1 mM EDTA, 10% Glycerol, 1% Triton X-100). Cell lysates were used for immunoprecipitation with anti-Flag antibody (1 µg per 500 µg of total proteins). The ubiquitination level of Flag-PP2A was detected through western blotting. For the deubiquitination assay, Flag-PP2A, HA-Ub (K48 or K63)and Myc-USP7 were transfected into 293T cells. To confirm whether proteasome-dependent degradation of substrates, cells were treated with 10 mM concentration of MG132 [a proteasome inhibitor, cat. no. F1100; Ubiquitin Proteasome Biotechnologies, LLC (UBPbio)] for 4 h before harvest and cultured at 37°C in a 5% CO₂ incubator. The cells were harvested after 48 h and immunoprecipitation was performed with anti-Flag antibody. Both assays were performed using an ubiquitination assay kit according to the manufacturer's manual (cat. no. UBAK-100; D&P Biotech Inc.; http://www.dnpbiotech.com).

GST-USP7 was transformed into the BL21 strain. After IPTG treatment, GST-USP7-transformed cells were cultured in an 18°C shaking incubator. The cells were then precipitated and released with phosphate-buffered saline (PBS) containing Triton X-100, PIC, and PMSF and then the cells were lysed using a sonicator. The Flag-PP2A-transfected 293T cell lysates (2 mg) were incubated with GST-USP7-fastened Glutathione Sepharose beads (300 µg per 2 mg of total protein; cat. no. GE17-0756-01; GE Healthcare; Cytiva). The beads were then washed with a lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, 150 mM NaCl, 1% Triton X-100). After removing the lysis buffer, 35 µl of 2X SDS buffer was added and boiled at 100°C for 7 min. The supernatants (25 µl) were then loaded onto 8% SDS-PAGE gel. The remaining steps were the same as those described for the western blotting. A portion of the sample was confirmed for protein expression through western blotting, and the expression of GST-USP7 using the remaining samples was confirmed through Coomassie Brilliant Blue (CBB) staining. The gels (1.5 mm thickness) were stained with 0.1% Coomassie Brilliant blue G 250 (cat. no. 1.15444; Sigma-Aldrich; Merck KGaA) at room temperature for 15 min. Subsequently, the gels were washed by destaining buffer (40% methanol and 10% acetic acid). Gel images were captured using a DUALED Blue/White Transilluminator (cat. no. A6020; Bioneer Corporation). Western blotting was performed to validate the results.

HeLa, MCF-7, and A549 cells were plated on a sterile glass and were washed with PBS. The cells (80% confluence) were treated with 4% paraformaldehyde at room temperature for 10 min and were then treated with 0.2% Triton X-100 in PBS at room temperature for 1 min. Cells were washed with PBS and were blocked in 1% bovine serum albumin (BSA) in PBS for 1 h. The samples were incubated with primary antibodies, PP2A (1:200) and USP7 (1:200; cat. no. CSB-PA849973LA01HU), at 4°C overnight. Cells were washed with PBS including 0.5% Tween-20 at room temperature for 10 min and were incubated with Alexa-Fluor-488-conjugated goat anti-mouse (cat. no. A32723; 1:500) and with Alexa-Fluor-568-conjugated goat anti-rabbit (cat. no. A11011; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Following incubation, nuclei were stained using DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 10 min. Cell images were obtained by a confocal microscope (Zeiss LSM880; Carl Zeiss Microscopy GmbH).

The sequences of small interfering RNA negative control (siNC) and siUSP7 were 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-CAUGCACAAGCAGUGCUGAAGAUAA-3′, respectively. siNC or siUSP7 was transfected into HeLa cells, and then cells were cultured in a 60-mm dish at 37°C in a 5% CO₂ incubator for 24 h. Transfection with siUSP7 was performed as previously described (27). After 24 h, cycloheximide (CHX) (cat. no. 01810; Sigma-Aldrich; Merck KGaA) was added to a final concentration of 100 µg/ml in siUSP7-transfected cells. Next, the cells were harvested in a time-dependent manner (0, 12 and 24 h) and cell lysates were used for western blotting.

For all measured data, the values for all samples obtained from at least three independent experiments were averaged, and the standard deviation or standard error was subsequently calculated. Statistical analysis was performed using the unpaired t-test and one-way analysis of variance followed by Tukey's multiple comparisons post hoc tests using GraphPad Prism version 5 (GraphPad Software, Inc.). Densitometric analysis was conducted using ImageJ software (version 1.4.3; National Institutes of Health).

In a previous study, 2-DE and MALDI-TOF/MS analysis determined that PP2A is a putative substrate of USP7 (17), thereby suggesting the possibility that PP2A could physically bind to USP7. To confirm the interaction between PP2A and USP7, a binding assay using 293T cells was performed. The results revealed binding between PP2A and USP7 (Fig. 1A), indicating the formation of an interaction between endogenous or exogenous PP2A and USP7. Additionally, in order to investigate the binding between these two proteins, the binding of PP2A was investigated with the expression of the differential amount of Myc-USP7. As a result, it was determined that as the concentration of Myc-USP7 increased, the amount of Myc-USP7 binding to PP2A also increased (Fig. 1B). Thereafter, the GST pull-down assay was performed to identify the occurrence of direct binding between the two proteins. GST-USP7 was incubated with overexpressed Flag-PP2A in 293T cell lysates, and subsequently subjected to Western blotting, which revealed that the two proteins bind directly each other (Fig. 1C). Furthermore, immunocytochemical analysis revealed that USP7 and PP2A are co-localized in the cytoplasm and the nucleus in HeLa cells, MCF-7 cells, and A549 cells. (Fig. 1D). Collectively, these results demonstrated the direct and indirect interplay between USP7 and PP2A in the cytoplasm and the nucleus.

The ubiquitination assay was performed to investigate whether PP2A is regulated by the UPS (Fig. 2A). Results obtained indicated that PP2A is degraded by the ubiquitin-mediated proteasomal degradation. The PTMs involve neddylation, SUMOylation, and ISGylation as well as ubiquitination (28). Moreover, numerous proteins undergo a proteasome-dependent degradation mechanism through ubiquitination, neddylation, SUMOylation, and ISGlyation (29). First, when MG132 was treated with the differential concentration, neddylation of Flag-PP2A was not increased at 2 and 5 µM of MG132 (Fig. 2B); a minimum 10-µM concentration was required to confirm the efficacy of MG132 on neddylation of PP2A. To assume that PP2A would also be modulated by conjugation with ubiquitin as well as ubiquitin-like molecules, IP was performed using co-transfected HA-neuronal precursor cell-expressed developmentally down-regulated protein 8 (HA-NEDD8) and Flag-PP2A in 293T cells with a proteasome inhibitor, MG132 (Fig. 2C), and it was determined that Flag-PP2A formed neddylation. However, SUMOylation and ISGylation on PP2A were not formed (data not shown). These results suggested that PP2A could also degrade via neddylation as well as ubiquitination due to increased expression level of neddylation on PP2A with the treatment of MG132. Therefore, PP2A could be regulated by PTMs including ubiquitination and neddylation. Increased levels of ubiquitinated or neddylated PP2A were observed after exposure to MG132, indicating that PP2A is degraded in a ubiquitination- or neddylation-mediated proteasome dependent manner. In addition, the ubiquitination assay of PP2A was performed with a K48-linked or K63-linked polyubiquitin chain (Fig. 2D). It was demonstrated that PP2A formed the K48-linked and K63-linked polyubiquitin chains. This suggested that PP2A may be affected by proteasome-dependent degradation associated with K48-linked polyubiquitin chain and intracellular effect by K63-linked ubiquitination (4). K63-linked polyubiquitin chains are known to regulate the proteasome-independent pathways such as signal transduction and endocytosis (5). By contrast, K48-linked polyubiquitin chains are known to regulate the proteasome-dependent pathway (5) Since PP2A formed a K63-linked polyubiquitin chain as shown in Fig. 2D, it is possible that the K63-linked polyubiquitination of PP2A is not related to proteasomal degradation. Furthermore, the reciprocal co-immunoprecipitation assays were performed as ubiquitination assays, as shown in Fig. S1. These results indicated that Flag-PP2A is connected to the HA-tagged polyubiquitin chain or polyneddylated chain.

To identify whether USP7 acts as a DUB for PP2A, a deubiquitination assay was performed subsequent to the expression of Myc-USP7, Flag-PP2A, and HA-Ub in 293T cells. USP7 decreased the ubiquitination level of PP2A, and inhibition of the enzymatic activity of USP7 exerted no change in the ubiquitination level of PP2A (Fig. 3A). These results indicated that the catalytic activity of USP7 induced the deubiquitination of PP2A. In addition, the effect of USP7 on the K48-linked or K63-linked polyubiquitin chain on PP2A was investigated (Fig. 3B). It was determined that USP7 deubiquitinated the K48-linked polyubiquitin chain on PP2A, with no associated difference in the K63-linked ubiquitination level on PP2A. This result indicated that USP7 deubiquitinated the K48-linked polyubiquitin chain on PP2A, thereby establishing that modulation of proteolysis is proteasome-dependent.

Since USP7 affects the protein stability by deubiquitinating the K48-linked polyubiquitin chain on PP2A, the protein levels of PP2A were investigated to determine whether USP7 controls the PP2A stability in a dose-dependent manner of siUSP7. First, the siUSP7 was used as previously determined (Fig. 4A) (27); the experimental protocol to confirm a decrease in the expression of USP7 was performed as previously described (Fig. 4B) (27). The expression of PP2A was dose-dependently reduced after exposure to varying concentrations of siUSP7 (Fig. 4C). Since the expression of PP2A was decreased subsequent to inhibition of USP7, it was further investigated whether siUSP7 also affects the ubiquitination of PP2A (Fig. 4D). The results confirmed that a dose-dependent increase in the ubiquitination level of PP2A subsequent to siUSP7 exposure indicated that the absence of USP7 may fail to induce deubiquitination of PP2A. Collectively, these experimental results revealed that the deubiquitination of PP2A was suppressed following inhibition of USP7, and PP2A stability was reduced by UPS. Conversely, overexpression of USP7 had no effect on the expression of PP2A (Fig. 4E); if the protein expression is too strong, the band is affected by saturation (30,31). Therefore, it appears that saturation of the PP2A protein signal in the western blotting may hinder the ability to observe an increase in the expression level of PP2A induced by USP7. Furthermore, the half-life of PP2A tended to be modulated by siUSP7 in a time-dependent manner of CHX exposure (Fig. 4F). This indicated that inhibition of USP7 reduced the protein stability of PP2A.