myc-usp10  (Boston Biochem)

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

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity

doi: 10.1016/j.jbc.2021.100405

Figure Lengend Snippet: CRISPR-Cas9 KO screen for ubiquitination-related proteins that regulate LC3B levels. A , workflow of CRISPR-Cas9 screen with a ubiquitination sgRNA library. For details of the screen, see . B , FACS profiles showing the enrichment of a population of H4-tfLC3B cells with low levels of GFP and mCherry signals from 0.6% to 90.8% after three rounds of selection and propagation. C , ranking of genes from the CRISPR-Cas9 screen based on the Robust Ranking Aggregation (RRA) score calculated using the MAGeCK algorithm. The deubiquitinating enzyme USP10 is indicated with a green dot . See Tables S1 and for additional information on the top hits.

Article Snippet: Plasmids encoding GFP-LC3B , FLAG-LC3B ( ) and FLAG-LC3B-K51R ( ) were described previously. pQCXIP-MYC-USP10 and pQCXIP-FLAG-USP10 were generated by subcloning coding sequences for human USP10 from FLAG-HA-USP10 into pQCXIP vector (TaKaRa, S3145) with an N-terminal MYC tag or FLAG tag.

Techniques: CRISPR, Selection

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity

doi: 10.1016/j.jbc.2021.100405

Figure Lengend Snippet: Silencing USP10 reduces the levels of both LC3B-I and LC3B-II. A , H4 cells were transfected with control or USP10 SMARTpool siRNAs. After 48 h, cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. Positions of the I and II forms of LC3B are shown with arrowheads . B , quantification of the ratio of LC3B-I and LC3B-II to β-tubulin. The LC3B-I to β-tubulin ratio for control siRNA transfection was arbitrarily set at 1. Bars represent the mean ± SD from three independent experiments such as that shown in A . Individual values from each experiment are represented by dots . The indicated p -value relative to LC3B-I in the control was calculated using a one-sample t -test; the p -value relative to LC3B-II in the control was calculated using an unpaired Student’s t -test. C , SDS-PAGE and immunoblot analysis of WT H4 cells and two clones of USP10-KO H4 cells using antibodies to the indicated proteins. D , quantification of the ratio of LC3B-I and LC3B-II to β-tubulin from three independent experiments such as that shown in C . Calculations and statistics were done as described in B . E , WT, USP10-KO, and MYC-USP10-rescued USP10-KO H4 cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. F , quantification of the ratio of LC3B-I and LC3B-II to β-tubulin from three independent experiments such as that shown in E . Calculations and statistics were done as described in B . In A , C , and E , the positions of molecular mass markers (in kDa) are indicated on the left.

Article Snippet: Plasmids encoding GFP-LC3B , FLAG-LC3B ( ) and FLAG-LC3B-K51R ( ) were described previously. pQCXIP-MYC-USP10 and pQCXIP-FLAG-USP10 were generated by subcloning coding sequences for human USP10 from FLAG-HA-USP10 into pQCXIP vector (TaKaRa, S3145) with an N-terminal MYC tag or FLAG tag.

Techniques: Transfection, SDS Page, Western Blot, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity

doi: 10.1016/j.jbc.2021.100405

Figure Lengend Snippet: Reduced LC3B-II levels in USP10-KO cells under conditions of bafilomycin A 1 treatment and starvation. A , WT and USP10-KO H4 cells were incubated with 50 nM bafilomycin A 1 (Baf A 1 ), starvation medium (Starv), or a combination of both for 2 h. Cells were then analyzed by SDS-PAGE and immunoblotting with antibodies to LC3B and β-tubulin. FM stands for fed medium (complete medium). B and C , quantification of the ratio of LC3B-I ( B ) and LC3B-II ( C ) to β-tubulin. The ratio for WT H4 cells in FM was arbitrarily set at 1. Bars represent the mean ± SD from three independent experiments such as that shown in A . Individual values from each experiment are represented by dots . The indicated p -value relative to WT H4 cells in the FM condition was calculated using a one-sample t -test; other p -values were calculated using an unpaired Student’s t -test. D , WT and USP10-KO H4 cells were incubated with 0, 20, or 50 μM MG132 for 6 h and analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. The accumulation of polyubiquitinated proteins ( middle panel ) was examined as a control for MG132 activity. E and F , quantification of the ratio of LC3B-I ( E ) and LC3B-II ( F ) to β-tubulin. The ratio for WT H4 cells without MG132 was arbitrarily set at 1. Bars represent the mean ± SD from three independent experiments such as that shown in D . Individual values from each experiment are represented by dots . The indicated p -values relative to WT H4 cells in the absence of MG132 were calculated using a one-sample t -test; other p -values were calculated using an unpaired Student’s t -test. In A and D , the positions of molecular mass markers (in kDa) are indicated on the left.

Article Snippet: Plasmids encoding GFP-LC3B , FLAG-LC3B ( ) and FLAG-LC3B-K51R ( ) were described previously. pQCXIP-MYC-USP10 and pQCXIP-FLAG-USP10 were generated by subcloning coding sequences for human USP10 from FLAG-HA-USP10 into pQCXIP vector (TaKaRa, S3145) with an N-terminal MYC tag or FLAG tag.

Techniques: Incubation, SDS Page, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity

doi: 10.1016/j.jbc.2021.100405

Figure Lengend Snippet: USP10 deubiquitinates LC3B. A , WT and USP10-KO H4 cells were transfected with plasmids encoding FLAG-LC3B or FLAG-LC3B-K51R, and HA-Ub. After 24 h, cells were incubated with 5 μM MG132 for 18 h. Cell lysates were immunoprecipitated with antibody to the FLAG epitope. Cell lysates and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with antibodies to FLAG and HA epitopes. The asterisk indicates the specific ubiquitinated LC3B band. Notice that USP10 KO increases the levels of ubiquitinated LC3B, but that under conditions of LC3B overexpression, it does not decrease total LC3B levels, probably because LC3B overexpression overwhelms the regulatory effect of ubiquitination. B , quantification of levels of ubiquitinated LC3B (immunoblotting with anti-HA antibody) normalized to nonubiquitinated LC3B (immunoblotting with anti-FLAG antibody). The relative level in WT H4 cells was arbitrarily set at 1. Bars represent the mean ± SD from three independent experiments such as that shown in A . Individual values from each experiment are represented by dots . The indicated p -value was calculated using a one-sample t -test. C , WT H4 cells were transfected with plasmids encoding FLAG-LC3B and FLAG-LC3B-K51R, and HA-Ub. Cell lysates were analyzed by immunoprecipitation with antibody to the FLAG epitope. The FLAG-LC3B bound to beads was incubated with recombinant His6-USP10 or His6-USP13, and the deubiquitination reaction mixtures were collected for further analysis. FLAG-LC3B was eluted from beads with 3xFLAG peptide. Cell lysates, elution products, and deubiquitination reaction mixtures were analyzed by SDS-PAGE and immunoblotting with antibodies to the FLAG, HA, and His6 epitopes. The asterisk indicates ubiquitinated LC3B. D , quantification of relative levels of ubiquitinated LC3B normalized to nonubiquitinated LC3B. The relative level for control without deubiquitinating enzymes was arbitrarily set at 1. Bars represent the mean ± SD from three independent experiments such as that shown in C . Individual values from each experiment are represented by dots . The indicated p -values were calculated using a one-sample t -test. E , WT H4 cells were transfected with plasmids encoding FLAG-USP10, FLAG-USP13, GFP-LC3B, and GFP. Cell lysates were immunoprecipitated with antibody to the FLAG epitope. Cell lysates and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with antibodies to GFP and FLAG epitope. In A , C , and E , the positions of molecular mass markers (in kDa) are indicated on the left.

Article Snippet: Plasmids encoding GFP-LC3B , FLAG-LC3B ( ) and FLAG-LC3B-K51R ( ) were described previously. pQCXIP-MYC-USP10 and pQCXIP-FLAG-USP10 were generated by subcloning coding sequences for human USP10 from FLAG-HA-USP10 into pQCXIP vector (TaKaRa, S3145) with an N-terminal MYC tag or FLAG tag.

Techniques: Transfection, Incubation, Immunoprecipitation, FLAG-tag, SDS Page, Western Blot, Over Expression, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity

doi: 10.1016/j.jbc.2021.100405

Figure Lengend Snippet: USP10 KO increases the accumulation of autophagy receptors and puromycin-induced aggregates. A , WT and USP10-KO H4 cells were incubated with 100 μg/ml cycloheximide (CHX) for 0, 2, 4, or 6 h and analyzed by SDS-PAGE and immunoblotting for SQSTM1, NBR1 and β-actin. The positions of molecular mass markers (in kDa) are indicated on the left. B and C , quantification of the ratio of SQSTM1 ( B ) and NBR1 ( C ) to β-actin at different time points. Values for WT H4 cells at time 0 were arbitrarily set to 1 and represent the mean ± SD from three independent experiments such as that shown in A . Individual values from each experiment are represented by dots . The indicated p -values were calculated using an unpaired Student’s t -test. D , confocal microscopy of WT and USP10-KO H4 cells incubated for 2 h without (control) or with 5 μg/ml puromycin, prior to immunostaining for SQSTM1 and Ub. Scale bar: 20 μm. Cells containing aggresome-like induced structures (ALIS) are indicated with arrowheads . The right-most column shows fivefold enlargements of the boxed areas. E , quantification of the percentage of ALIS-containing WT and USP10-KO H4 cells treated with 5 μg/ml puromycin for 2 or 3 h from experiments such as that shown in D . Bars represent the mean ± SD of the percentages of cells with ALIS from four independent experiments counting more than 1200 cells. Individual values from each experiment are represented by dots . The indicated p -values were calculated using an unpaired Student’s t -test. F , schematic representation of the LC3B ubiquitination/deubiquitination cycle driven by UBA6-BIRC6 and USP10.

Article Snippet: Plasmids encoding GFP-LC3B , FLAG-LC3B ( ) and FLAG-LC3B-K51R ( ) were described previously. pQCXIP-MYC-USP10 and pQCXIP-FLAG-USP10 were generated by subcloning coding sequences for human USP10 from FLAG-HA-USP10 into pQCXIP vector (TaKaRa, S3145) with an N-terminal MYC tag or FLAG tag.

Techniques: Incubation, SDS Page, Western Blot, Confocal Microscopy, Immunostaining