2.1 Clinical samples
Breast cancer specimens and paired adjacent normal breast tissue specimens were obtained from ten patients with primary breast cancer. All patients received no therapy before sample collection. The study protocol was approved by an independent ethics committee of Fourth Military Medical University. The study was undertaken in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki. All patients were asked to provide written informed consent before enrollment. All tissues were collected immediately upon resection and transported in liquid nitrogen.
2.2 Cells, antibodies and reagents
Human breast cancer cell lines (MDA-MB-231, MCF-7) were obtained from ATCC. MDA-MB-231 cells were cultured in DMEM medium supplemented with 10% FBS. MCF-7 cells were grown in DMEM medium supplemented with 10% and 0.01 mg/ml bovine insulin. All the cells were cultured in a humidifed atmosphere with 5% CO2 at 37 °C.
2.3 TCGA database analyses
Clinical relevance of USP41 in breast cancer was analyzed based on the public database of TCGA. A total of 1104 cancer and 113 normal specimens were included to examine USP41 expression. A total of 541 USP41 high expression patients and 541 USP41 low expression patients were included to analyze overall survival (http://starbase.sysu.edu.cn/panGeneDiffExp.php#，Flold Changes=5.02，p Value=4.8*10E14).
2.4 Cell proliferation analysis
CCK-8 assay was performed to measure proliferation of breast cancer cells according to manufacturer’s instructions. Briefly, 6000 cells/well were seeded in 96-well plates in medium containing 10% FBS and incubated under 37 ℃，5% CO2. After treatment, 10 ul CCK-8 reagent was added and incubated for 1 hour, the absorbance under 450 nm was measured with a microplate reader. The same experiments were repeated after a defined incubation period.
2.5 Colony formation assay
The different cells were seeded in 6-cm dishes at a density of 300 cells/dish. Following incubation for 2 weeks in a humidified incubator at 37˚C in an atmosphere of 95% air and 5% CO2, the cells were washed with phosphate-buffered saline (PBS), and colonies were fixed with methanol for 10 min and stained with 0.5% crystal violet for 15 min. The number of colonies was counted under a microscope (D750; Nikon, Tokyo, Japan). All experiments were performed in triplicate dishes in 3 independent experiments.
Transwell migration assay was performed using transwell inserts. 8 × 104 cells in serum-free medium were seeded into the upper chamber of the insert and the bottom of the chamber contained the DMEM medium with 10% FBS. After 36 h incubation, the cells were fixed with methanol and stained with Giemsa. Then cells on the top surface of the membrane were wiped off, and cells on the lower surface were examined under an inverted light microscope. The number of migrated or invaded cells was quantified by counting the number of cells from 10 random fields at ×100 magnification.
Additionally, cellular apoptosis was determined by flow cytometry (FAC). Cell lines cultured in DMEM supplemented with 10% FCS were seeded in 96-well plates (2 × 104 cells/well). After treatment with USP41 overexpression or knockdown, cellular apoptosis was determined by FAC after a stain with Annexin v-FITC or Propidium Iodide (PI) or both for 15 min in the dark at room temperature following the instruction of Annexin
v-FITC apoptosis detection kit (YEASEN, China).
2.8 Cell cycle analysis
Cell cycle distribution was analyzed using ﬂow cytometric analysis. Brieﬂy, after treatment with USP41 overexpression or knockdown, cells were harvested and fixed with precooled 75% (v/v) ethanol for 24h. Afterwards, ethanol was discarded by centrifugation. Fibroblasts were rehydrated with PBS at room temperature and resuspended in propidium iodide (PI) DNA staining buffer and incubated for 30 min at room temperature in the dark. Detection was performed on a flow cytometer (FACSAria; BD Biosciences).
2.9 RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from the cells and tissues using Trizol reagent (Invitrogen, USA), according to the manufacturer's instructions. cDNA was generated from 1 µg total RNA using SuperScript III (Invitrogen, USA) and polyN primers. qPCRwas performed using the ABI 7500 fast system (Thermofisher, USA) with the primers as follow: USP41, 5'-TGGAGGGCAGTATGAGCTTTTT-3' (Forward Primer) and 5'-ATGACCGGAGTCTGCCATTC-3' (Reverse Primer); RACK1, 5'-CCACCACGAGGCGATTTGT-3' (Forward Primer) and 5'-CCCAGGGTATTCCATAGCTTGAT-3' (Reverse Primer). The relative levels of gene expression were represented as 2 - Δ Ct (Ct gene - Ct reference) . The experiments were repeated in triplicate.
2.10 USP41 overexpression and knockdown
USP41 overexpression lentivirus and small-interfering RNA (siRNA) were purchased from Beijing Syngentech Cooperation (Beijing, China) and HIPPOBIO (Wuhan, China), respectively. MCF7 and MDA-MB-231 cells were transfected with lentiviruses for 72 h. qPCR and western blot were used to verify the efficacy of USP41 knockdown. Then, USP41-overexpression and USP41-knockdown breast cancer cells were used for function detection.
2.11 Western blot
Cells were homogenized and lysed with RIPA lysis buffer (100 mM NaCl, 50 mM Tris–HCl pH 7.5, 1% TritonX-100, 1 mM EDTA, 10 mM b-glycerophosphate, 2 mMsodium vanadate and protease inhibitor). Protein concentration was assayed using the micro-BCA protein assay (Pierce, Rockford, IL). 40 lg of protein per lane was separated by 12% SDS-PAGE and electroblotted onto nitrocellulose (Amersham Pharmacia, Germany). Then, the membrane was blocked with 5% non-fat milk and incubated with antibodies against 4EBP1 (Cell Signaling, USA, 1:1000), P-4EBP1 (Cell Signaling, USA, 1:1000), Ps6 (Cell Signaling, USA, 1:1000),β-cantenin (Servicebio, China, 1:1000), Cleaved-caspase 3 (Cell Signaling, USA, 1:1000), CyclinD1 (Santa Cruz, USA, 1:1000), CyclinE (Santa Cruz, USA, 1:1000), FASN (Cell Signaling, USA, 1:1000), M-TOR (Cell Signaling, USA, 1:1000), PM-TOR(Cell Signaling, USA, 1:1000), p-cjun (Cell Signaling, USA, 1:1000), USP41 (Invitrogen, USA, 1:1000), P-P38 (Cell Signaling, USA, 1:1000), P53 (Santa Cruz, USA, 1:1000), PTEN (Cell Signaling, USA, 1:1000); E-cad (Cell Signaling, USA, 1:1000), HKII (Cell Signaling, USA, 1:1000), PSTAT3 (Cell Signaling, USA, 1:1000), N-Cad (Cell Signaling, USA, 1:1000), P21 (Cell Signaling, USA, 1:1000), PAKT (Cell Signaling, USA, 1:1000), P-bad (Cell Signaling, USA, 1:1000), PKM2 (Cell Signaling, USA, 1:1000), RACK1 (Proteintech, USA, 1:1000),β-actin (Servicebio, China, 1:1000), and GAPDH (Proteintech, USA, 1:1000). Incubation with the primary antibody was carried out overnight in a cold room. The membrane was then incubated with a secondary antibody conjugated to goat anti-mouse IgG (1:5 000, sigma) and developed using enhanced chemiluminescence (ECL, Amersham Pharmacia, NJ).
2.12 CoIP-MS lysed by adding protease inhibitor to RIPA lysate
CoIP-MS was used to explore the interacting proteins with USP41. The process consists of 5 main steps: (1) incubation of cell lysates with Flag-tag antibodies (Sigma-Aldrich, USA), following cell transfection with Flag-USP41 and cell lysis with RIPA lysate, (2) binding of immune complexes to protein A/G agarose beads (Sigma-Aldrich, USA), (3) removal of non-interacting proteins, (4) elution to obtain protein interacting complexes, (5)mass spectrometry identification of protein interacting complexes. The enriched co-immunoprecipitation products were analyzed by mass spectrometry. Peptides with scores b20 were removed, and higher scores meant a better degree of matching with the secondary atlas. Peptides were searched and compared qualitatively in UniPro. The UniquePep Count and the Cover Percent were also evaluated as auxiliary metrics for the final identification results.
2.13 Statistical analysis
The data are presented as the means ± SD. Student’s t test was used for statistical analysis, unless otherwise indicated. GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA) and SPSS 23.0 Software (IBM Inc., Armonk, NY) were used for all statistical analyses. P-values <0.05 were considered to indicate statistically significant differences.