Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

doi: 10.1016/j.jbc.2021.101088

Figure Lengend Snippet: USP10 inhibits AKT phosphorylation and deubiquitinates PTEN with K63-linked polyubiquitination. A , HEK293T cells were transfected with a collection of 48 Dubs for 48 h. IB assays were performed to measure AKT phosphorylation level and total AKT expression. B , the ratio of p-AKT/AKT in the presence of each Dub (1, USP49; 2, USP28; 3, USP26; 4, USP15; 5, ATXN3; 6, USP14; 7, JOSD2; 8, USP29; 9, CYLD; 10, USPL1; 11, JOSD3; 12, USP53; 13, USP25; 14, USP38; 15, USP52; 16, USP45; 17, OTUD4; 18, A20; 19, USP43; 20, USP22; 21, USP21; 22, USP16; 23, PARP11; 24, USP20; 25, OTUD7B; 26, JOD1; 27, USP42; 28, USP44; 29, JOSD1; 30, USP36; 31, BAP1; 32, empty vector; 33, UCHL1; 34, USP3; 35, USP30; 36, UCHL3; 37, USP48; 38, USP1; 39, USP7; 40, UCHL5; 41, USP5; 42, USP50; 43, USP39; 44, USP8; 45, USP46; 46, USP33; 47, USP10; 48, USP13; and 49, USP11). C , A549 cells were cotransfected with K63-Ub and selected Dubs for 48 h, followed by IP/IB assay as indicated. D , A549 cells were transfected with USP10 plasmids for 48 h, followed by IP/IB assays. E , USP10 was knockdown by siRNA from H1299 cells for 48 h, followed by IP/IB assays. F , UPS10 was knocked out from H1299 cells by using its sgRNA species. Seventy-two hours later, cells were subjected to IP/IB assays against K63-linked polyubiquitination. G , Flag-PTEN (F-PTEN) purified from K63-Ub–expressing cells were incubated with ATP and purified USP10 in tube for 30 min. After termination, the reaction was subjected to IP/IB assays to measure PTEN ubiquitination. H , A549 and H1299 cells were transfected with the Myc-tagged WT (M-WT) or its C424A mutant USP10 for 48 h, followed by IP/IB assays as indicated. Dub, deubiquitinase; HEK293T, human embryonic kidney 293T; IB, immunoblotting; IP, immunoprecipitation; PTEN, phosphatase and tensin homolog deleted on chromosome 10; sgRNA, single-guide RNA; USP10, ubiquitin-specific protease 10.

Article Snippet: In the study of USP10 removing TRIM25-mediated Ub chains from PTEN, purified Flag-PTEN, Flag-TRIM25, and Myc-USP10 were added to the reaction mixture containing ATP, HA-Ub, with or without E1 and E2 (Boston Biochem).

Techniques: Transfection, Expressing, Plasmid Preparation, Purification, Incubation, Mutagenesis, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

doi: 10.1016/j.jbc.2021.101088

Figure Lengend Snippet: USP10 deubiquitinates PTEN in K63-linked polyubiquitination mediated by TRIM25. A , A549 cells were cotransfected with TRIM25 and selected deubiquitinases based on Figure 1 and literatures for 48 h, followed by IP/IB assay as indicated. B , HEK293T cells were cotransfected with PTEN, TRIM25, K63-Ub, and USP10 or ATXN3 for 48 h, followed by IP/IB assays against specific antibodies as indicated. C , HEK293T cells were cotransfected with plasmids of PTEN, TRIM25, K63-Ub, and wtUPS10 or C424A mutant or USP7 (a negative control) for 48 h, followed by IP/IB assays. D , purified E1, E2, ATP, Ub, PTEN as well as TRIM25 and USP10 were incubated in tube for 30 min. After termination, the reaction was subjected to IB assay for PTEN ubiquitination assay. E , WT PTEN or its K266R mutant was cotransfected with K63-Ub and USP10 plasmids for 48 h, followed by IP/IB assays as indicated. HEK293T, human embryonic kidney 293T; IB, immunoblotting; IP, immunoprecipitation; PTEN, phosphatase and tensin homolog deleted on chromosome 10; TRIM25, tripartite motif containing 25; USP10, ubiquitin-specific protease 10.

Article Snippet: In the study of USP10 removing TRIM25-mediated Ub chains from PTEN, purified Flag-PTEN, Flag-TRIM25, and Myc-USP10 were added to the reaction mixture containing ATP, HA-Ub, with or without E1 and E2 (Boston Biochem).

Techniques: Mutagenesis, Negative Control, Purification, Incubation, Ubiquitin Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

doi: 10.1016/j.jbc.2021.101088

Figure Lengend Snippet: USP10 competitively binds to PTEN against TRIM25. A , HEK293T cells were cotransfected with Flag-PTEN and Myc-USP10 plasmids for 48 h, followed by IP/IB assays. B , A549 and H1299 cell lysates were subjected to IP and IB as indicated. C , A549 and H1299 cells were subjected to immunofluoresence assay as indicated. D , HEK293T cells were transfected with USP10 and TRIM25 plasmids for 48 h, followed by IP/IB assays as indicated. E , H1299 cell lysates were subjected to reciprocal IP/IB assays with specific antibodies as indicated. F , A549 and H1299 cells were transfected with a Myc-USP10 (M-USP10) plasmid for 48 h, followed by IP/IB assays as indicated. HEK293T, human embryonic kidney 293T; IB, immunoblotting; IP, immunoprecipitation; PTEN, phosphatase and tensin homolog deleted on chromosome 10; TRIM25, tripartite motif containing 25; USP10, USP10, ubiquitin-specific protease 10.

Article Snippet: In the study of USP10 removing TRIM25-mediated Ub chains from PTEN, purified Flag-PTEN, Flag-TRIM25, and Myc-USP10 were added to the reaction mixture containing ATP, HA-Ub, with or without E1 and E2 (Boston Biochem).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

doi: 10.1016/j.jbc.2021.101088

Figure Lengend Snippet: The stability of PTEN protein is not affected by USP10. A , HEK293T cells were cotransfected with Flag-PTEN and Myc-USP10 plasmids as indicated for 48 h, followed by IB assay. B , HEK293T cells were transfected with USP10 and PTEN plasmids for indicated periods, followed by IB assay. C and D , after being transfected with USP10 plasmids for 48 h, A549 and H1299 cells were applied for RT-PCR assay to measure PTEN mRNA level ( C ) and IB to measure its protein level ( D ). E , HEK293T cells were transfected with a USP10 plasmid for 24 h, followed by cycloheximide (CHX) treatment for indicated periods. Cell lysates were then prepared for IB assay. F , the statistical analysis of PTEN stability based on E . G , USP10 was knocked out from H1299 cells by its specific sgRNA species, 48 h later, cells were treated with CHX for indicated periods, followed by cell lysate preparation and IB assay. H , the statistical analysis of PTEN stability based on G . HEK293T, human embryonic kidney 293T; IB, immunoblotting; PTEN, phosphatase and tensin homolog deleted on chromosome 10; sgRNA, single-guide RNA; USP10, ubiquitin-specific protease 10.

Article Snippet: In the study of USP10 removing TRIM25-mediated Ub chains from PTEN, purified Flag-PTEN, Flag-TRIM25, and Myc-USP10 were added to the reaction mixture containing ATP, HA-Ub, with or without E1 and E2 (Boston Biochem).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

doi: 10.1016/j.jbc.2021.101088

Figure Lengend Snippet: USP10 restores PTEN activity and suppresses activation of the AKT/mTOR signaling pathway. A , the USP10 expression profile was evaluated by IB in various cell lines. B , primary NSCLC (C) and paracancerous (P) tissues were subjected to IB assay for USP10 and TRIM25 expressions. C , the statistical analysis of USP10 and TRIM25 in primary NSCLC tissues based on B . D , A549 and H1299 cells were transfected with increasing Myc-USP10 plasmids for 48 h, followed by IB assay against specific antibodies as indicated. E , USP10 was knocked out by its specific sgRNA, 72 h later, cells were subjected to IB assay. F , A549 and H1299 cells were treated with spautin-1 for 24 h, and cell lysates were subjected to IB assay. G , PTEN was knocked down by its specific shRNA from A549 and H1299 cells, followed by transfection of USP10 plasmids. Cells were then subjected to IB assay as indicated. H , A549 and H1299 cells were transfected with USP10 plasmids for 48 h, followed by the measurement of PI(3,4,5)P3. ∗∗∗ p < 0.01. I , WT or K266R mutant PTEN was transfected into A549 and H1299 cells along with Myc-TRIM25 or Myc-USP10. About 24 h later, cells were subjected to isolation of plasma membrane (PM) from the cytosol fraction. The individual fractions were then subjected to IB for indicated proteins. IB, immunoblotting; mTOR, mammalian target of rapamycin; NSCLC, non–small cell lung cancer; PI(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; sgRNA, single-guide RNA; TRIM25, tripartite motif containing 25; USP10, ubiquitin-specific protease 10.

Article Snippet: In the study of USP10 removing TRIM25-mediated Ub chains from PTEN, purified Flag-PTEN, Flag-TRIM25, and Myc-USP10 were added to the reaction mixture containing ATP, HA-Ub, with or without E1 and E2 (Boston Biochem).

Techniques: Activity Assay, Activation Assay, Expressing, Transfection, shRNA, Mutagenesis, Isolation, Membrane, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

doi: 10.1016/j.jbc.2021.101088

Figure Lengend Snippet: USP10 suppresses NSCLC cell proliferation and migration. A , A549 and H1299 cells transfected with a USP10 plasmid were replated in 96-well plates for MTT assay. B , A549 and H1299 cells were transfected with a USP10 plasmid for 48 h, followed by cell cycle analyses. C , A549 and H1299 cells were transfected with a USP10 plasmid for 48 h, followed by EdU incorporation and fluorescent microscopy assay. D , the statistical analysis of EdU-positive cells from C . E , USP10 was knocked out by its sgRNA from A549 and H1299 cells. Seventy-two hours later, cells were incorporated with EdU and analyzed by fluorescent microscopy assay. F , the statistical analysis of EdU-positive cells from E . G , A549 and H1299 cells were transfected with a WT or C424A mutant USP10 plasmid for 48 h, followed by transwell assay. H , A549 and H1299 cells were subjected to knock out USP10 using sgRNA, followed by transwell assay. EdU, 5-ethynyl-2′-deoxyuridine; MTT, 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; NSCLC, non–small cell lung cancer; sgRNA, single-guide RNA; USP10, ubiquitin-specific protease 10.

Article Snippet: In the study of USP10 removing TRIM25-mediated Ub chains from PTEN, purified Flag-PTEN, Flag-TRIM25, and Myc-USP10 were added to the reaction mixture containing ATP, HA-Ub, with or without E1 and E2 (Boston Biochem).

Techniques: Migration, Transfection, Plasmid Preparation, MTT Assay, Microscopy, Mutagenesis, Transwell Assay, Knock-Out