Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: USP22 regulates the secretion of EVs. A) Schematic representation of the experimental design of the xenograft model (n = 6 mice per group). B–D) Tumor images (B), growth weights (C), and proliferation curves (D). E) USP22 was analyzed using western blotting of the xenograft tumors. F) HE, and IHC staining of Ki67 and USP22. Scale bars, 25 µm. G) Heatmap of the differences between mice with USP22‐Ctrl and USP22 knockdown after proteomic sequencing. H,I) GO (H) and KEGG (I) analysis based on the differential genes of the xenograft tumors. J,K) Characterization of serum‐derived EVs using TEM (J), western blotting analysis (K) of CD9, CD63, TSG101 (EV‐derived protein markers), and USP22. Scale bars, 200 nm. L) NTA analysis of the effect of USP22 on EV secretion in vivo. M) Relative mRNA expression of USP22 in serum‐derived EVs of healthy individuals and patients with LUAD detected using qRT‐PCR (n = 12). N) GO analysis of differentially expressed proteins in USP22 knockdown and control H1299 cells. O) NTA results of EVs revealing the total number of particles isolated from each group. P) Confocal microscopy analysis of CD63 in LUAD lines. Scale bars, 20 µm. Q CD63 expression of EVs isolated from the same culture media (CM) and cell membrane proteins were tested using western blotting. Data are presented as mean ± SD. Statistical significance was determined by Student's t ‐test and ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Western Blot, Immunohistochemistry, Knockdown, Sequencing, Derivative Assay, In Vivo, Expressing, Quantitative RT-PCR, Control, Isolation, Confocal Microscopy, Membrane

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: USP22 regulates EV secretion from LUAD cell lines via stabilizing MYO1B. A) MS identification of MYO1B as a potential USP22 interacting protein in H1299 cells. B) Visualization of USP22‐MYO1B binding. C) Co‐IP assays revealing the interaction between USP22 and MYO1B in H1299 cells. D) IF imaging shows the colocalization of USP22 and MYO1B in LUAD cells. Scale bars, 20 µm. E) PLA assay was performed in shCtrl and shUSP22 H1299 cells: each PLA signal represents a molecular interaction between USP22 and MYO1B. Scale bars, 10 µm. F) Western blotting of MYO1B protein expression in LUAD cell lines with USP22 knockdown and overexpression. G) qRT‐PCR of MYO1B expression in LUAD cell lines with USP22 knockdown and overexpression. H) Western blotting of MYO1B expression treated with CHX (200 µg mL −1 ). I) Western blotting of MYO1B expression treated with MG132 (10 µ m ) for 6 h. J) Impact of USP22 knockdown and overexpression on MYO1B ubiquitination. K) Schematic representation of the structures of USP22 and its truncated mutants. L) MYO1B interactions with USP22 and its mutants. M) Determination of MYO1B ubiquitination via co‐IP and western blotting analysis of MYO1B using an anti‐ubiquitin antibody. N) Schematic representation of USP22 and its point mutants. O) MYO1B interactions with USP22 and its point mutants. P) USP22 wild type or point mutants were transfected to measure MYO1B ubiquitination in H1299 cells. Q) MYO1B and USP22 protein levels were detected in H1299 cells treated with CHX. R) H1299 cells were co‐transfected with HA‐Ub WT, or HA‐K6‐Ub (Lys6‐only), or HA‐K11‐Ub (Lys11‐only), or HA‐K27‐Ub (Lys27‐only), or HA‐K29‐Ub (Lys29‐only), or HA‐K33‐Ub (Lys33‐only), or HA‐K48‐Ub (Lys48‐only), or HA‐K63‐Ub (Lys63‐only) plasmids, and then the MYO1B ubiquitylation linkage was detected using IP analysis with anti‐HA antibody after treatment with MG132 for 6 h. Data are presented as mean ± SD. Statistical significance was determined by Student's t ‐test. Not significant (ns), p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Imaging, Western Blot, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Transfection

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: EV‐derived USP22 isolated from LUAD cells enhances tumor cell motility. A) Volcano plot of differentially expressed proteins of shCtrl EVs and shUSP22 EVs in H1299 cells obtained via proteomic analysis. B) KEGG enrichment analysis. C) GO enrichment analysis. D) Cell migration and invasion abilities of LUAD cells co‐cultured with EVs were examined using transwell assay. Scale bars, 100 µm. E) Cell migration ability of LUAD cells co‐cultured with EVs were examined via wound healing assay. Scale bars, 200 µm. F) LUADs treated with EVs were stained with invadopodia markers, cortactin and F‐actin (Phalloidin). Scale bars, 20 µm. G) In vitro metastasis model. H) Trans‐endothelial migration and intravasation/extravasation. Scale bars, 250 µm. I) Western blotting analysis of the effect of EVs on the expression of malignant proteins in LUAD cells. Data are presented as mean ± SD. Statistical significance was determined by ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Derivative Assay, Isolation, Migration, Cell Culture, Transwell Assay, Wound Healing Assay, Staining, In Vitro, Western Blot, Expressing

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: EV‐derived USP22 promotes tumor growth in xenograft tumors. A) Schematic diagram of the xenograft tumor model. (n = 6 mice per group). B–D) Tumor images (B), growth weights (C), and proliferation curves (D). E) Western blotting analysis of the expression of USP22, E‐cadherin, N‐cadherin, vimentin, MMP2, MMP9, MMP14, and PCNA. F) IF images of invadopodia in tumor tissues. G) HE and IHC staining of USP22, Ki67, E‐cadherin, N‐cadherin, MMP2, MMP9, MMP14, CD31, and CD63. Scale bars, 25 µm. Data are presented as mean ± SD. Statistical significance was determined by ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Derivative Assay, Western Blot, Expressing, Immunohistochemistry

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: USP22 interaction with KDELR1, and USP22 effects on KDELR1 protein stabilization and ubiquitination. A) DEGs were identified after USP22 knockdown in H1975 cells. B) GO analysis of DEGs. C) Visualization of USP22‐KDELR1 binding. D) Interaction between USP22 and KDELR1 in H1299 cells verified by co‐IP assay. E) IF staining was used to examine the colocalization of USP22 and KDELR1 in LUAD cells. Scale bars, 20 µm. F) Representative images of PLA signals between USP22 and KDELR1. Scale bars, 10μm. G) Protein expression of KDELR1 in LUAD cells with USP22 knockdown and overexpression. H) mRNA expression of KDELR1 in LUAD cells with USP22 knockdown and overexpression. I) Western blotting analysis of KDELR1 treated with CHX (200 µg mL −1 ). J) Western blotting analysis of KDELR1 treated with MG132 (10 μ m ) for 6 h. K) Impact of USP22 knockdown and overexpression on KDELR1 ubiquitination were examined. L) Identification of the binding domains of USP22 and KDELR1. M) Effects of USP22 and its mutants on KDELR1 ubiquitination were detected via co‐IP assay. N) KDELR1 interactions with USP22 and its point mutants were examined. O) Effects of USP22 and its point mutants on KDELR1 ubiquitination were analyzed. P) Protein stability of KDELR1 was analyzed after transfection with USP22 and its point mutant plasmids. Q) KDELR1 ubiquitination assays in H1299 cells co‐transfected with HA‐Ub WT, or HA‐K6‐Ub, or HA‐K11‐Ub, or HA‐K27‐Ub, or HA‐K29‐Ub, or HA‐K33‐Ub, or HA‐K48‐Ub, or HA‐K63‐Ub plasmids after treatment with MG132 for 6h. Data are presented as mean ± SD. Statistical significance was determined by Student's t ‐test. Not significant (ns), p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Knockdown, Binding Assay, Co-Immunoprecipitation Assay, Staining, Expressing, Over Expression, Western Blot, Transfection, Mutagenesis

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: USP22 promotes the KDELR1/SRC pathway. A) Western blotting analysis of SRC cascade expression after Dasatinib treatment of USP22‐knockdown or USP22‐overexpressing cells. B) KDELR1 knockdown attenuated USP22‐mediated SRC signaling activation, whereas KDELR1 overexpression had the opposite effect. C) Invadopodia were visualized by colocalization of cortactin and F‐actin (Phalloidin). Scale bars, 20 µm. D) Trans‐endothelial migration and intravasation/extravasation by in vitro metastasis model. Scale bars, 250 µm. E) Schematic diagram of siRNA treatment. (n = 5 mice per group). F–H) In vivo analysis of tumor (F), weight (G), and proliferation curves (H) in xenograft tumors after intra‐tumoral injection of in vivo‐optimized KDELR1 inhibitor or the control twice a week. I) IF images of tissues stained with cortactin and F‐actin. Scale bars, 50 µm. J) HE and IHC staining images. Scale bars, 50 µm. Data are presented as mean ± SD. Statistical significance was determined by Student's t ‐test and ANOVA test. Not significant (ns), p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Western Blot, Expressing, Knockdown, Activation Assay, Over Expression, Migration, In Vitro, In Vivo, Injection, Control, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: Antitumor effect of 13‐MB on LUAD in vitro and in vivo. A) Effect of different concentrations of 13‐MB after 24, 48, and 72 h on the survival fraction of LUAD cells. B,C) Effect of 13‐MB (20 and 40 µ m ) on the proliferative capacity of LUAD cells as detected by clone formation (B) and EdU assays (C). D) Results of transwell assay for detecting the migratory and invasive abilities of cells treated with 13‐MB. E) Apoptosis of LUAD cells treated with different concentrations of 13‐MB for 24 h, which were stained with fluorescein isothiocyanate (FITC)‐conjugated Annexin V antibody and propidium iodide (PI) before flow cytometry. F) LUAD cells were treated with different concentrations of 13‐MB for 24 h. Western blotting analysis of the protein expression levels of USP22, caspase‐3, cleaved caspase‐3, caspase‐9, cleaved caspase‐9, PARP, and cleaved PARP. G) Schematic diagram of the effects of 13‐MB in vivo. (n = 5 mice per group). H–J) Tumor images (H), growth weights (I), and proliferation curves (J). K) Western blotting analysis of the expression of USP22 and apoptosis indicators in tumor tissues. L) Tumor tissues were stained for USP22, Ki67, cleaved caspase‐3, cleaved caspase‐9, and cleaved PARP using HE and IHC staining. Scale bars, 25 µm. Data are presented as mean ± SD. Statistical significance was determined by Student's t ‐test and ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: In Vitro, In Vivo, Transwell Assay, Staining, Flow Cytometry, Western Blot, Expressing, Immunohistochemistry

Journal: Advanced Science

Article Title: Ubiquitin‐Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma

doi: 10.1002/advs.202405731

Figure Lengend Snippet: The mechanisms of USP22 mediates tumor cell‐derived EV secretion to promote tumor motility.

Article Snippet: Lentivirus vectors with UPS22 overexpression and USP22 shRNA were purchased from GeneChem.

Techniques: Derivative Assay

Journal: Cancer research

Article Title: USP22 functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA repair

doi: 10.1158/0008-5472.CAN-19-1033

Figure Lengend Snippet: (A) Female heterozygous mice with human USP22 in the Rosa26 locus (Rosa26-Lox-Stop-Lox(3XFLAGUSP22)) were crossed with male prostate-specific probasin cre recombinase mice (PB-Cre4) to produce offspring with no expression of hUSP22 (WT) or with expression of hUSP22 (PbCre/USP22). Mice were aged at least 12 months and their prostate lobes were micro-dissected to detect expression of hUSP22 RNA (WT N=9; Pb-Cre/USP22 N=11). Student’s t-test. (B) The prostate lobes of WT and PbCre/USP22 mice aged at least 12 months underwent IHC analysis for Ki67. Representative images are shown at 40X and quantitative analysis are shown to the right (WT N=9; Pb-Cre/USP22 N=10). Student’s t-test. (C) Mouse adult fibroblasts (MAFs) from a male heterozygous mouse with human USP22 in the Rosa26 locus (Rosa26-Lox-Stop-Lox(3XFLAGUSP22)) were immortalized and then transduced with GFP control (GFP) or cre recombinase (hUSP22) to induce expression of hUSP22. (D) GFP or hUSP22 MAFs were plated for proliferation or survival assays and were treated with vehicle (N=5) or 5Gy irradiation (N=4) and measured using pico green fluorescence on Day 0, 3, and 6. Survival assays were plated and then treated on Day 0 with the designated dose of IR (N=6). (E) GFP or hUSP22 MAFs were plated for survival (N=3) or caspase-3 activity assays/western blot and treated with the indicated dose of cisplatin on Day 0. Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: SMARTvector Human Inducible non-targeting mCMV-TurboGFP control shRNA or USP22 shRNA (Dharmacon) were used for inducible knockdown of USP22.

Techniques: Expressing, Transduction, Irradiation, Fluorescence, Activity Assay, Western Blot

Journal: Cancer research

Article Title: USP22 functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA repair

doi: 10.1158/0008-5472.CAN-19-1033

Figure Lengend Snippet: (A) RNA-seq analysis was performed in LN-pLPLUC and LN-USP22hi cells, as well as LN-shCon and LN-shUSP22–4693 cells after 5 days of 1 μg/mL doxycycline treatment in biological triplicate, as shown in the PCA analysis. Cells were treated as described and immunoblotted with the indicated antisera. (B) MA plots show differential gene expression in LN-USP22hi cells compared to LN-pLPLUC (Left; Blue) and LN-shUSP22–4693 cells compared to LN-shCon (Right; Green). (C) Using normalized RNA-seq count data, GSEA was used to identify MSigDB pathways that were enriched upon USP22 depletion and de-enriched upon USP22 overexpression. A p value of <0.05 was used to determine significant enrichment. *represents cell cycle or DNA repair-related pathways. (D) Cells were plated for proliferation assays and treated with vehicle (UT) or irradiation (IR; LN-shCon and LN-shUSP22–4693 +2Gy IR (N=5); C42-shCon and C42-shUSP22–4693 +5Gy IR (N=3)). Cell numbers were measured using pico green fluorescence on Day 0, 3, and 6. Cells were treated with 1 μg/mL doxycycline every 48 hours. Relative cell number was achieved by normalizing to Day 0. 2-way ANOVA. (E) C42-shCon or C42-shUSP22–4693 cells were plated for clonogenic survival assays and treated with IR or cisplatin at the designated doses. Cells were treated with 1 μg/mL doxycycline every 2–3 days. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: SMARTvector Human Inducible non-targeting mCMV-TurboGFP control shRNA or USP22 shRNA (Dharmacon) were used for inducible knockdown of USP22.

Techniques: RNA Sequencing Assay, Expressing, Over Expression, Irradiation, Fluorescence

Journal: Cancer research

Article Title: USP22 functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA repair

doi: 10.1158/0008-5472.CAN-19-1033

Figure Lengend Snippet: (A) Alterations, defined as amplification, homozygous deletion, mutation, mRNA upregulation, mRNA downregulation, or fusion, in USP22 occur in both primary and metastatic prostate adenocarcinoma from studies available in the cBioportal (Excluded are those studies with <1% alterations in USP22: Broad-Cornell 2013; CPC-GENE 2017; MSKCC-DFCI 2018); Studies with * represent those studies that include only genomic alterations (amplification, homozygous deletion, or mutation). (B) USP22 gene expression is positively correlated with AR or MYC gene expression using the cBioportal TCGA provisional prostate adenocarcinoma study (N=498). Inset represents co-occurrence (p value, Fisher Exact Test) of alterations in USP22 with MYC or AR. (C) Amplification and/or mRNA upregulation of USP22 are associated with decreased progression free survival in the TCGA provisional dataset (p=0.0459) using all complete tumors and the MSKCC Cancer Cell dataset (p=0.0342) using all complete tumors. Log rank test. (D) Using the MSKCC Cancer Cell 2010 dataset (cBioportal), USP22 expression was measured as a function of Gleason Grade. (E) USP22 alterations are most commonly amplification events or mRNA upregulation events. Left, those studies with only genomic alterations available (See * in Figure 1A). Right, those studies with both genomic and RNA alterations available. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: SMARTvector Human Inducible non-targeting mCMV-TurboGFP control shRNA or USP22 shRNA (Dharmacon) were used for inducible knockdown of USP22.

Techniques: Amplification, Mutagenesis, Expressing

Journal: Cancer research

Article Title: USP22 functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA repair

doi: 10.1158/0008-5472.CAN-19-1033

Figure Lengend Snippet: (A) Ubiscan workflow and models used for analyses. LNCaP parental cells were transfected with shUSP22 or USP22. Three samples (LNCaP: parental, shUSP22, and USP22) were run as duplicate injections for a total of 6 LC-MS/MS experiments. (B) Differential ubiquitylation of histones H2A and H2B upon USP22 knockdown and overexpression compared to LNCaP parental control. (C) Ubiscan analysis demonstrates differentially ubiquitylated peptides upon USP22 overexpression and USP22 knockdown. Putative targets of USP22 differential ubiquitination designated as >1.5-fold decreased peptide ubiquitylation in LN-USP22 compared to control and >1.5-fold increased peptide ubiquitylation in LN-shUSP22 compared to parental control (Peptides denoted in purple). (D) Putative direct targets of USP22 function as determined by (C) with DNA repair-related peptides designated by inclusion in gene set enrichment analysis pathways (KEGG or Hallmark GSEA).

Article Snippet: SMARTvector Human Inducible non-targeting mCMV-TurboGFP control shRNA or USP22 shRNA (Dharmacon) were used for inducible knockdown of USP22.

Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy, Over Expression

Journal: Cancer research

Article Title: USP22 functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA repair

doi: 10.1158/0008-5472.CAN-19-1033

Figure Lengend Snippet: (A) Cell lysates underwent immunoprecipitation of USP22 and immunoblot analysis of XPC in LN-pLPLUC and LN-USP22hi (left), C42-pLPLUC and C42-USP22hi (middle), and GFP and hUSP22 MAFs (right). (B) Indicated cells grown in full serum were isolated for immunoblot analysis with the designated antisera. (C) Cell lysates underwent Ubitest analysis (Life Sensors) with pulldown of poly-ubiquitylated proteins, then +/− deubiquitylase (DUB) treatment, and subsequent immunoblot analysis of XPC in LN-pLPLUC and LN-USP22hi (left) and C42-pLPLUC and C42-USP22hi (right). The change in XPC levels was compared +/− DUB relative to input. The blots are a representative image from three independent experiments. (Student t-test; N=3). (D) LN-pLPLUC or LN-USP22hi cells underwent immunoblot analysis or proliferation assays 72 hours post-transfection of siControl or siXPC +/− 2Gy irradiation (N=3). Student’s t-test. (E) LN-pLPLUC and LN-USP22hi (left) and C42-pLPLUC and C42-USP22hi (right) cells were used for immunoblot analysis of chromatin tethering experiments. (F) LN-pLPLUC or LN-USP22hi, C42-pLPLUC or C42-USP22hi, LN-shCon or LN-shUSP22–4693, and C42-shCon or C42-shUSP22–4693 cells were plated for immunofluorescence analysis of XPC. A representative experiment is shown and at least three independent experiments were performed. Student’s t-test. (G) LN-pLPLUC or LN-USP22hi, C42-pLPLUC or C42-USP22hi, LN-shCon or LN-shUSP22–4693, and C42-shCon or C42-shUSP22–4693 cells were plated and immunofluorescence of XPC was analyzed 24 hours post-5Gy irradiation. A representative experiment is shown and at least three independent experiments were performed. Student’s t-test. (H) LN-shCon or shUSP22–4693 cells were treated with 10 J UV irradiation (N=3 independent experiments in technical triplicate) and cyclobutane pyrimidine dimers (CPDs) were measured by ELISA. (I) Model of study findings. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: SMARTvector Human Inducible non-targeting mCMV-TurboGFP control shRNA or USP22 shRNA (Dharmacon) were used for inducible knockdown of USP22.

Techniques: Immunoprecipitation, Western Blot, Isolation, Transfection, Irradiation, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Oncology Research

Article Title: Downregulation of Ubiquitin-Specific Protease 22 Inhibits Proliferation, Invasion, and Epithelial–Mesenchymal Transition in Osteosarcoma Cells

doi: 10.3727/096504016X14772395226335

Figure Lengend Snippet: USP22 was upregulated in OS tissues and cell lines. (A, B) The mRNA and protein expression levels of USP22 were markedly increased in OS tissues compared with the corresponding normal tissues. (C, D) The expression of USP22 was much higher in the OS cell lines U2OS and MG-63 than in the osteoblastic cell line hFOB at both mRNA and protein levels. * p < 0.05.

Article Snippet: USP22 siRNA was purchased from RiboBio (Guangzhou, P.R.

Techniques: Expressing

Journal: Oncology Research

Article Title: Downregulation of Ubiquitin-Specific Protease 22 Inhibits Proliferation, Invasion, and Epithelial–Mesenchymal Transition in Osteosarcoma Cells

doi: 10.3727/096504016X14772395226335

Figure Lengend Snippet: Downregulation of USP22 inhibited OS cell proliferation and invasion in vitro. (A, B) USP22 downregulation in U2OS and MG-63 cells was confirmed by Western blot analysis. (C, D) USP22 downregulation significantly inhibited the proliferative ability of U2OS and MG-63 cells. (E, F) USP22 downregulation obviously reduced the number of invading U2OS and MG-63 cells. * p < 0.05.

Article Snippet: USP22 siRNA was purchased from RiboBio (Guangzhou, P.R.

Techniques: In Vitro, Western Blot

Journal: Oncology Research

Article Title: Downregulation of Ubiquitin-Specific Protease 22 Inhibits Proliferation, Invasion, and Epithelial–Mesenchymal Transition in Osteosarcoma Cells

doi: 10.3727/096504016X14772395226335

Figure Lengend Snippet: Downregulation of USP22 inhibited OS tumor growth and metastasis in vivo. (A, B) USP22 downregulation significantly decreased the tumor volume and weight of the U2OS/USP22si group compared with the U2OS/NCsi group. (C) The number of lung nodules was obviously reduced in the U2OS/USP22si group compared with the U2OS/NCsi group. * p < 0.05.

Article Snippet: USP22 siRNA was purchased from RiboBio (Guangzhou, P.R.

Techniques: In Vivo

Journal: Oncology Research

Article Title: Downregulation of Ubiquitin-Specific Protease 22 Inhibits Proliferation, Invasion, and Epithelial–Mesenchymal Transition in Osteosarcoma Cells

doi: 10.3727/096504016X14772395226335

Figure Lengend Snippet: Downregulation of USP22 inhibited the EMT process in OS cells. (A) The protein expression levels of EMT-related markers in U2OS cells were detected by Western blot. (B) The protein expression of EMT-related markers in U2OS cells was quantified by an Odyssey infrared laser imaging system. * p < 0.05.

Article Snippet: USP22 siRNA was purchased from RiboBio (Guangzhou, P.R.

Techniques: Expressing, Western Blot, Imaging

Journal: Oncology Research

Article Title: Downregulation of Ubiquitin-Specific Protease 22 Inhibits Proliferation, Invasion, and Epithelial–Mesenchymal Transition in Osteosarcoma Cells

doi: 10.3727/096504016X14772395226335

Figure Lengend Snippet: Downregulation of USP22 inhibited the activation of the PI3K/Akt signaling pathway. (A) The protein expression levels of p-PI3K, PI3K, p-Akt, and Akt in U2OS cells were detected by Western blot. (B) U2OS and MG-63 cells were incubated with different concentrations of MK-2206. Cell growth was determined after 24 h of using MTT assay. (C, D) U2OS and MG-63 cells were transfected with USP22si or NCsi in the presence or absence of MK-2206 (100 nM). The Transwell assay was carried out to measure cell invasion. * p < 0.05.

Article Snippet: USP22 siRNA was purchased from RiboBio (Guangzhou, P.R.

Techniques: Activation Assay, Expressing, Western Blot, Incubation, MTT Assay, Transfection, Transwell Assay