Cell culture and transfection

HEK293 (KCLB: 21573), HeLa (KCLB:10002), A549 (KCLB: 10185) and H1299 (KCLB: 25803) cells were maintained in DMEM (Gibco BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (Gibco) at 37 °C in a humidified atmosphere with 5% CO2. The cells were passaged every 3–4 days. Cells at 60–80% confluence were transfected with the indicated constructs using polyethyleimine (PEI) (Polysciences, Inc. Cat no. 24765), Lipofectamine 3000 (Cat no. L3000001, Thermo Fisher Scientific) or Lipofectamine 2000 (Cat no. 11668019 Life Technologies), following the manufacturers’ instructions. The cells were harvested 36–48 h post transfection for further analysis.

Plasmids and sgRNAs

The Myc-MAST1 plasmid was generated as described previously [21]. Briefly, the human MAST1 gene was amplified from cDNA and cloned into the pCDNA3 6XMyc-vector using BamHI and XbaI restriction sites. Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene. The catalytic mutant of USP28 was generated using site-directed mutagenesis by substituting cysteine with alanine at position 171; the resultant vector was named Flag-USP28C171A. The plasmid encoding Cas9-2a-mRFP-2a-PAC (puromycin N-acetyl-transferase puromycin resistance gene) and plasmid encoding sgRNAs were purchased from Toolgen (Seoul, South Korea). To generate sgRNAs targeting potential DUB candidates, sgRNA target sequences were designed using a public tool (www.broadinstitute.org) and cloned into vectors as described previously [26]. Briefly, oligonucleotides containing the USP28 target sequence were synthesized (Bioneer, Seoul, South Korea), and T4 polynucleotide kinase was used to add terminal phosphates to the annealed oligonucleotides (Bio-Rad, CA, USA). The vector was digested using BsaI restriction enzyme and ligated with the annealed oligonucleotides. The target sequences for the sgRNAs targeting USP28 are listed in Supplementary Table S1.

Antibodies and reagents

Mouse monoclonal antibodies against Flag (Anti-DDDDK-tag, M185-3L, 1:1,000) were purchased from MBL Life Science, and phospho-Histone H2AX (Ser139) (Merck, 05-636) was purchased from Millipore. Mouse monoclonal antibodies against MAST1 (sc-373845, 1:50), H2AX (sc-517336; 1:1000), c-Myc (SC-40, 1:1,000), ubiquitin (sc-8017, 1:1,000), HA (sc-7392, 1:1,000), GAPDH (sc-32233, 1:1000), normal mouse IgG (sc-2025, 1:1000), MEK1 (sc-219, 1: 1000), ERK1/2 (sc-514302, 1:1000), p-ERK 1/2 (sc-81492, 1:1000), and BIM (H5) (sc-374358, 1:1000) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against MAST1 (Cusabio CSB-PA897529LA01HU, 1:1000), USP28 (Proteintech 17707-1-AP, 1:1500) and 488/594-conjugated secondary antibodies (Cat. no. A21207 and Cat. no. A21203, 1:200; Life Technologies) were used. p-MEK1 (Cat no. 9121, 1: 1000) and cleaved PARP (D64E10, 1: 1000) were purchased from Cell Signaling Technology.

IP lysis buffer (Cat. no. 87787; Thermo Fisher), cell lysis buffer (Cat. no. R2002, Biosesang), protein 5X sample buffer (Cat. no. EBA-1052, Elpis Biotech), Protein A/G Plus agarose beads (sc-2003, Santa Cruz Biotechnology), protease inhibitor cocktail (Cat. no. 11836153001, Roche), the protein translation inhibitor cycloheximide (CHX; Cat. no. 239765, Merck), the proteasomal inhibitor MG132 (Cat. no. S2619, Selleckchem), puromycin (Cat. no. 12122530, Gibco), cisplatin (Cat no. P4394, Sigma-Aldrich), the DUB inhibitor PR-619 (ab144641, Abcam), AZ1 USP25/28 inhibitor (Cat. no. 7845, Tocris), CCK-8 assay reagent (Dojindo Molecular Technologies, MD, USA) and DAPI (Cat. no. H-1200, Vector Laboratories) were purchased and used.

Cell viability assay for dose response curve

The relative viability of the cells post cisplatin treatment was measured using a Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan). Cells were seeded in 96-well plates at density of 1 × 104 s per well and were grown for 24 h. The cells were treated with increasing concentration of cisplatin for 48 h. Later, 10 μL of CCK-8 assay solution was added and incubated for 4 h. Absorbance was recorded at 450 nm using spectrophotometer (Bio-Rad Laboratories, Inc, Korea). IC50 and IC80 value of cisplatin in HeLa (IC50 = 6.3 µg/mL and IC80–15 µg/mL), A549 (IC50 = 3.5 µg/mL and IC80–13 µg/mL) and H1299 (IC50 = 6.6 µg/mL and IC80–19.5 µg/mL) cells were calculated.

Generation of cisplatin resistance HeLa cell line

HeLa Cisplatin-resistant cells (HeLa-cisR) were derived from their respective parental cell lines by gradual exposure to cisplatin which is dissolved in 0.9% Saline (Sigma-Aldrich, UK). Briefly, the HeLa cells were seeded at a density of 1 × 106 and subjected to stepwise increases upto 15 μg of cisplatin/mL over a period of 6 months. The cisR cell lines were grown as monolayer cultures and maintained in the DMEM medium containing cisplatin (5 μg/mL) and supplemented with 10% FBS (Gibco) and 1% penicillin and streptomycin (Gibco) at 37 °C in a humidified atmosphere with 5% CO2.

T7 endonuclease 1 assay

Genomic DNA was isolated using DNeasy Blood & Tissue kits (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The region of DNA containing the nuclease target site was PCR-amplified and denatured by heating and annealed to form heteroduplex DNA, which was then treated with 5 units of T7E1 (New England Biolabs, MA, USA) for 15 to 20 min at 37 °C, followed by 2% agarose gel electrophoresis. Mutation frequencies were calculated based on band intensity using ImageJ software and the following equation: mutation frequency (%) = 100 × (1 − [1 − fraction cleaved]1/2), where the fraction cleaved was the total relative density of the cleavage bands divided by the sum of the relative density of the cleaved and uncut bands. The oligonucleotide sequences used for PCR amplification for the T7E1 assay are listed in Supplementary Table S2. The PCR-amplicon sizes of the USP28 gene and the expected cleavage sizes after the T7E1 assay are summarized in Supplementary Table S3.

Real-time PCR

Total RNA was isolated using Trizol reagent (Favorgen, Kaohsiung, Taiwan). The reverse transcription reaction was performed using a SuperScript III First-Strand Synthesis System (Life Technologies, USA) with an oligo-dT primer according to the manufacturer’s protocol. Quantitative PCR was performed in triplicate using Fast SYBR Green I Master Mix (Life Technologies) and a Step One Plus Real-Time PCR System (Life Technologies). The oligonucleotide sequences used for qRT-PCR are mentioned in Supplementary Table S4.

Generation of a USP28-knockout cell line using CRISPR/Cas9

The single cell–derived USP28 KO clones were generated using the CRISPR/Cas9 system as described previously [27, 28]. A549 and H1299 cells were co-transfected with a plasmid encoding Cas9 and sgRNA2 targeting USP28 or scrambled sgRNA (mock control) at a 1:2 ratio using Lipofectamine 3000 Reagent or Lipofectamine 2000 reagent, respectively, according to the manufacturer’s instructions. Later, the cells were selected using puromycin (1 µg/mL) for next 2 days. The puromycin-selected cells were seeded into 96-well plates at 25 cells/plate and incubated in a CO2 incubator at 37 °C. After 15 days, the wells were microscopically evaluated and the single cell–derived colonies were selected. The selected colonies were dissociated using trypsin–EDTA and reseeded into 24-well cell culture plates. A small portion of selected colonies was used to isolate genomic DNA and screened for USP28 disruption by T7E1 assay. The T7E1 positive single cell–derived clones were expanded and stored in a liquid nitrogen tank after Sanger sequencing. USP28 mRNA and protein levels in USP28-KO clones were determined by RT-PCR and western blotting, respectively. Cell lines showing complete reduction in USP28 mRNA and protein levels were used for in vitro and in vivo experiments.

Immunoprecipitation

Cells were transfected with the indicated DNA constructs. At 36–48 h post transfection, the cells were lysed in IP lysis buffer ((25 mM Tris–HCl (pH 7.4), 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 5% glycerol, 1 mM PMSF, and protease inhibitor cocktail) for 20 min and the amount of protein was estimated using Bradford reagent. Cell lysate (2–3 mg) was immunoprecipitated using the indicated antibodies at 4 °C overnight and then incubated with 35 μL of protein agarose beads at 4 °C for 3 h. The agarose beads were washed with lysis buffer and eluted in 2X SDS sample loading buffer (5X SDS sample loading buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris–HCl [pH 6]). The eluted samples were boiled at 95 °C–100 °C for 5 min and separated on SDS-PAGE gels by western blotting. Mouse IgG (ab-99697, 1: 10,000; Abcam) and rabbit IgG (CST-58802S, 1: 10,000; Cell Signaling Technology) light chain-specific secondary antibody was used to prevent interference from heavy and light immunoglobulin chains in the binding assay.

Tandem ubiquitin-binding entities assay

The ubiquitination status of MAST1 protein was determined using a tandem ubiquitin binding entities (TUBEs) assay (Cat. no. UM402, LifeSensors, PA, USA) as previously described [29]. The mock control, USP28-KO A549 and USP28-KO H1299 cells were pretreated with the proteasome inhibitor MG132 (10 μM/mL) for 6 h to accumulate polyubiquitinated MAST1 protein. Cells were lysed in IP lysis buffer containing 150 mM sodium chloride, 1% Triton X-100, 25 mM Tris (pH 7.5), 1 mM EDTA, 10% glycerol, and protease inhibitor cocktail. The lysed protein extracts were incubated with 20 µL of ubiquitin affinity matrices-TUBE2 at 4 °C for 3 h with rotation. The beads were washed with IP lysis buffer and samples were eluted in 30 µL 2X SDS sample loading buffer (5X SDS sample loading buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, 0.125 M Tris–HCl (pH 6.8)). The eluted samples were boiled at 95 °C–100 °C for 5 min, separated by SDS-PAGE and analyzed by western blotting.

Deubiquitination assay

The DUB activity of USP28 against endogenous and exogenous MAST1 protein was determined in A549 and HEK293 cells, respectively. The cells were treated with MG132 (10 µM/mL for 6 h) 48 h post transfection and harvested. The cells were lysed for 20 min in denaturing lysis buffer containing 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 50 mM Tris–HCl (pH 7.4), 2 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail. Cell lysates (2–3 mg) were immunoprecipitated with the respective antibodies at 4 °C overnight and incubated with 35 μL of protein agarose beads for 2–3 h at 4 °C. The agarose beads were washed with lysis buffer and samples were eluted in 2X SDS sample loading buffer (5X SDS sample loading buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris–HCl [pH 6]). The eluted samples were boiled at 95 °C–100 °C for 5 min, separated on SDS-PAGE gels and analyzed by western blotting using anti-ubiquitin and anti-HA antibodies. To avoid non-specific binding of polyubiquitin molecules to MAST1 protein, the protein-bound agarose beads were washed with lysis buffer containing 300 mM NaCl as previously described [30].

Immunofluorescence staining

A549 and H1299 cells were grown on glass coverslips and incubated at 37 °C in a humidified atmosphere with 5% CO2. The cells were washed with phosphate-buffered saline (PBS, Gibco), fixed for 15 min using 4% paraformaldehyde (PFA, Biosesang) and permeabilized in PBS containing 0.1% Triton X for 5 min at room temperature. The cells were washed, blocked in 3% bovine serum albumin and stained with indicated primary antibodies overnight at 4 °C. The next day, the cells were washed with PBS and incubated with Alexa Fluor 488–conjugated secondary antibodies for 1 h. The nuclei were stained with DAPI and cells were mounted using VectaShield (Vector Laboratories, CA, USA). The cells were then visualized and images were produced using a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Wetzlar, Germany).

Duolink proximity ligation assay (PLA)

The interaction between USP28 and MAST1 was observed using a Duolink in situ proximity ligation assay (PLA) kit (Cat. no. DUO92101, Sigma Aldrich) according to the manufacturer’s instructions. A549 cells were fixed in 4% PFA for 10 min at room temperature and then blocked with blocking solution. The cells were treated with primary antibodies targeting MAST1 and USP28 for 1 h at 37 °C, followed by incubation with PLA probes for 1 h at 37 °C in a humidified chamber. After three washes, ligation ligase solution was added, and the cells were incubated for 30 min at 37 °C. The slides were incubated for 100 min in an amplified polymerase solution at 37 °C in the dark. Finally, the cells were stained with mounting medium containing DAPI. A Leica fluorescence microscope was used to capture the fluorescence images (Leica, DM 5000B; Leica CTR 5000; Wetzlar, Germany).

Immunohistochemistry (IHC)

Clinical tissue microarray slides of lung, colon and breast tumors were purchased from ISU Abxis (Gyeonggi-do, South Korea). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were processed and incubated with USP28 or MAST1 antibody according to the supplier’s protocol. The samples were counterstained with hematoxylin, dehydrated, and mounted. The staining intensity was determined using ImageJ IHC profiler, an open source plugin for quantification and scoring of IHC images [31]. The staining was scored as 0 (no stain), + 1 (weak stain), + 2 (moderate stain) and + 3 (strong stain) based on the intensity of staining. The results of multiplying the percentage of cells with staining intensity values were added to calculate the H-score. The relationship between the protein expression level of USP28 and MAST1 in different tissues was analyzed using non-parametric Spearman correlation test in order to test the significance of combined tissue IHC-expression.

Tumor tissue xenografts obtained from mice were fixed with 4% PFA and embedded in paraffin. FFPE tissues were then sectioned at a thickness of 5 µm and stained with USP28 and MAST1 following the manufacturers’ recommendations. The mounted IHC tissue samples were visualized and images were produced using a Leica DM5000 B microscope (Leica, Germany).

Cell proliferation assay

A549 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) and H1299 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) were treated with either vehicle (saline) or cisplatin for 48 h. Next, 10 μL of CCK-8 assay reagent (Dojindo Molecular Technologies, MD, USA) was added to each well, and absorbance was measured at 450 nm using a spectrophotometer (Bio-Rad Laboratories, Inc., Korea). The concentrations of cisplatin used for A549 and H1299 cells were 2 μg/mL and 5 μg/mL, respectively.

Apoptosis assay

The Annexin-V/PI (Propidium Iodide) population was detected using a BD FACSCanto II flow cytometer (BD Biosciences, CA, USA). Briefly, A549 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) were treated with either vehicle (saline) or cisplatin for 48 h. The cells were then harvested and washed twice with PBS containing 10% FBS. The cells were counted, and 5 µL of Annexin-V and PI (BD Pharmingen™ FITC Annexin V apoptosis detection kit, Cat. no. 556547, BD biosciences, San Diego, CA, USA) were added to cells, followed by incubation for 15 min. The stained cells were resuspended in binding buffer, and flow cytometry was performed within 1 h.

Cell cycle analysis

Cell cycle analysis was performed by PI staining (BD Biosciences). A549 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) and H1299 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) were treated with vehicle or cisplatin for 48 h and then harvested, washed twice with ice-cold PBS containing 10% FBS, and fixed with ice-cold 70% ethanol. The cells were resuspended in PI (50 µg/mL; Sigma) and RNase A (200 µg/mL, New England Biolabs, MA, USA) and subjected to FACS analysis (BD FACSCanto II, BD Biosciences) to measure DNA content. Data were analyzed using FACS Diva software (version 8, BD bioscience). Next, 10 μL of CCK-8 assay reagent (Dojindo Molecular Technologies, MD, USA) was added to each well, and absorbance was measured at 450 nm using a spectrophotometer (Bio-Rad Laboratories). The concentrations of cisplatin used for A549 and H1299 cells were 2 μg/mL and 5 μg/mL, respectively.

Soft agar assay

A549 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) and H1299 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) were examined by colony formation assay. Firstly, 1% agarose gel and 1X complete DMEM were mixed at a ratio of 1:1 and plated onto 35 mm culture dishes. The plates were then incubated overnight. Cells resuspended in 0.75% agarose with DMEM (1:1 ratio) were seeded at a density of 1 × 104 cells per well. The cells were treated with vehicle or cisplatin every other day for 14 days. Crystal violet dye (0.01%) diluted in 20% methanol was used to stain the anchorage-independent colonies, and colonies were counted using a light microscope (IX71, Olympus, Tokyo, Japan).

Wound healing assay

A549 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) and H1299 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) were cultured to near 90% confluence. Scratches were made in the monolayers with a sterile pipette tip. The wounded cell layer was washed with PBS and plates were incubated in medium containing either vehicle or cisplatin at 37 °C with 5% CO2. Wound closure was compared at 0 h and 24 h post-scratch using a light microscope and quantified using ImageJ software.

Transwell cell invasion assay

Transwell chambers (0.8 µm pore) were coated with Matrigel for 1 h at 37 °C (Corning, NY, USA) according to the manufacturer’s instructions. A549 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) and H1299 cells (mock control, USP28KO, USP28KO-reconstituted with USP28 or MAST1) were seeded at a density of 3.0 × 104 cells per well in 500 µL of serum-free DMEM in the top chamber. Next, 750 µL of complete media containing either vehicle or cisplatin was added to the bottom chambers. Plates were then incubated at 37 °C with 5% CO2 for 24 h. Cells on the top surface of the insert were scraped off, and the cells on the bottom surface were fixed with ice-cold methanol followed by crystal violet staining. Cells were visualized and images were produced using a Leica DM5000 B microscope. The number of cells was counted using ImageJ, and the data are presented graphically.

Animal studies

The animal study was approved by the Institutional Animal Care and Use Committees of Hanyang University.

Study design: The four groups including Mock, USP28-KO, USP28-KO cells reconstituted with USP28 and USP28-KO cells reconstituted with MAST1.

Treatment strategy: Cisplatin (2 mg/kg) prepared in saline (Vehicle) was delivered by intraperitoneal injection (i.p.) twice a week for 14–16 days.

Animal species or strain: Mouse/ NOD scid gamma/ NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (005557).

Animal sex: 2 males and 2 females per group.

Age and weight: 6-week old mice weighing between 20 and 25 g.

Sample size: Four mice per group (n = 4).

Total number of animals used in this study: Total four groups, four mice per group (n = 4). Total 16 mice were used in this study.

Inclusion and exclusion: No mice were excluded from the study.

Randomization: The animal house staff with no prior knowledge of experiment to be performed randomly allocated age matched mice of equivalent genetic background for the study.

Blinding: The animal house staff allocated mice for the study. For this study, three different researchers were involved as follows: First leading investigator performed subcutaneous injection of samples from each group. Second leading investigator performed cisplatin treatment. Third leading investigator conducted euthanization and surgical procedure.

Drug: Cisplatin; Vehicle: Saline; Dose: Cisplatin (2 mg/kg); Cell lines: A549 cells.

Site and route of administration: Cell were subcutaneously injected into the right flank of each mice. Cisplatin was delivered by intraperitoneal injection (i.p.) twice a week for 14–16 days.

Detailed protocol: Mice were housed in a temperature-controlled room under standard conditions (12 h light/dark cycle at a temperature of 27 °C and 55% relative humidity) with access to food and water ad libitum. A549 cells (1.0 × 107) transfected with Mock control, USP28-KO, USP28-KO reconstituted with USP28 or MAST1 were prepared in DMEM: Matrigel (1:1) (BD Biosciences) and subcutaneously injected into the right flank of each mouse (four mice per group, n = 4). Cisplatin (2 mg/kg) was delivered by intraperitoneal injection (i.p.) twice a week from the day 7 till the end of the experiment. Mice were weighed two times a week and the experiment was terminated on the 27th day of the treatment. At the end of the study, all mice were euthanized. The tumors were harvested at the end of the experiment and images were taken.

Method of euthanasia: CO2 asphyxiation and tumors were collected by dissection.

Outcome measures: Tumor growth was recorded by measuring two perpendicular diameters (short axis and long axis) and tumor volume was calculated using the formula V = D × d2 × 0.5, where D and d are the long and short axes of the tumor, respectively. Tumor volume was measured every other day and is presented graphically. Mice were weighed two times a week and the experiment was terminated on the 27th day of the treatment.

Statistical methods: Statistical analysis and graphical presentation were performed using GraphPad Prism 9.0. Two-way ANOVA followed by Tukey's post-hoc test was used with the indicated P values. Statistical power is 80% and was calculated post experiment using G*Power software (https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower.html) [32].

Statistical analysis

Statistical analysis and graphical presentation were performed using GraphPad Prism 9.0. All results are presented as the means and standard deviations of at least three independent experiments (unless otherwise stated in the figure legends). The error bar represents means and standard deviations. Comparisons between two groups were analyzed using Student’s t-test. Experiments involving three or more groups were analyzed by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The relationship between the protein expression level of USP28 and MAST1 in different human tissues was determined by H-score and analyzed using non-parametric Spearman correlation test. P-values < 0.05 were regarded as statistically significant. IC50 and IC80 values of cisplatin in HeLa, A549 and H1299 cells were calculated by non-linear regression analysis.