Journal: bioRxiv

Article Title: Common molecular mechanisms underlie the transfer of alpha-synuclein, Tau and huntingtin and modulate spontaneous activity in neuronal cells

doi: 10.1101/2021.07.18.452825

Figure Lengend Snippet: (A) Immunoblots showing protein levels in cell lysates and released to the cell media of cells expressing the different proteins. HEK cell stably expressing 25QHtt-EGFP, 103QHtt-EGFP, aSyn-EGFP and EGFP-Tau were transfected with USP19 or with the catalytic inactive form USP19 C506S. Quantifications were normalized to total protein levels using MemCode. (B) LDH measurements confirm the absence of cell toxicity and cell death in the experiments. (C) Tau is more strongly internalized by naïve cells. Percentage of EGFP positive cells after incubation with media from cells co-expressing 25QHtt-EGFP, 103QHtt-EGFP, aSyn-EGFP or EGFP-Tau together with USP19 or USP19 C506S for 24 hours. Cell counting was performed using flow cytometry. Data from at least three independent experiments for each condition. Significant differences were assessed by one-way ANOVA followed by multiple comparisons with significance between groups corrected by Bonferroni procedure. Differences were considered to be significant for values of p<0.05 and are expressed as mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following plasmids used for the transfection protocol: pcDNA 3.1, pcDNA 3.1-aSyn, pcDNA 3.1-Tau (4R2N), pcDNA 3.1-22QHtt exon 1 (1-90, CAG, ID CHDI-90000027, Coriell Institute), pcDNA 3.1-72QHtt exon 1 (1-90, CAG, ID CHDI-90001882-1, Coriell Institute), mCitrine-USP19 (Plasmid #78593, Addgene), mCitrine-USP19 C506S (Plasmid #78594, Addgene).

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Incubation, Cell Counting, Flow Cytometry

Journal: Scientific Reports

Article Title: USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth

doi: 10.1038/s41598-018-37264-5

Figure Lengend Snippet: SiRNA screen identifies USP19 as a modulator of EWS-FLI1 stability. ( a ) In-silico selection of candidates. 21 deubiquitinating enzymes were selected based on their expression levels from publicly available microarray data sets of Ewing cell lines and tumors. ( b ) Screening setup. A673 and RDES cells stably expressing flag-tagged EWS-FLI1 were reverse transfected with single siRNAs from a small siRNA library. After 48 h, lysates were incubated in anti-flag coated plates to determine EWS-FLI1 protein normalized to total protein input. ( c ) EWS-FLI1 protein levels upon candidate knockdown. Each dot represents 3xflag-EWS-FLI1 protein levels normalized to its total protein for each single well. 3xflag-EWS-FLI1 levels upon USP19 knockdown are indicated with larger red dots and upon EWS-FLI1 knockdown in orange. ( d ) Expression levels of USP19 in indicated cell lines and primary samples were analyzed by western blot using USP19 antibody. The arrows indicate specific USP19 isoforms, asterisk marks unspecific band. ( e ) mRNA expression of USP19 was determined by quantitative RT-PCR from same cells and normalized to GAPDH.

Article Snippet: USP19 cDNA (Addgene #36306) was introduced into pCMV-3xflag or pCDNA-3xmyc vectors (tag N-terminal of USP19) by In-Fusion cloning HD (Clontech Laboratories Inc., Mountain View, CA, USA) according to manufacturer’s protocol.

Techniques: In Silico, Selection, Expressing, Microarray, Stable Transfection, Transfection, Incubation, Knockdown, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth

doi: 10.1038/s41598-018-37264-5

Figure Lengend Snippet: USP19 specifically modulates EWS-FLI1 protein levels. ( a , b ) Immunoblot analysis of USP19 depleted cells. ( a , b ) A673 and SKNMC cells were transiently transfected with 20 nM siRNAs for 72 h as indicated. Lysates were subjected to western blot analysis and analyzed by anti-FLI1, anti-USP19 and anti-p27 antibodies. Arrows indicate specific USP19 isoforms, asterisk marks an unspecific band. Right panel to each western blot, quantification of EWS-FLI1 proteins levels (n = 5–6, the mean is indicated by the horizontal line, error bars as SD). ( c ) Active USP19 stabilizes EWS-FLI1 protein. 3xflag-EWS-FLI1 was transiently co-expressed with a control vector or increasing levels (ratios 3xflag-EWS-FLI1 to 3xmyc-USP19 1:2 and 1:4) of wild-type or catalytically inactive 3xmyc-USP19 for 48 h in HEK293T cells. Lysates were analyzed by western blotting using anti-flag and anti-myc antibodies. ( d ) Quantification of 3xflag-EWS-FLI1 protein levels of ( c ) with n = 8 for control and n = 4 for others, the mean is indicated by the horizontal line and error bars as SD. ( e ) USP19 overexpression stabilizes specifically EWS-FLI1. 3xflag-EWS-FLI1, 3xflag-EWSR1 and 3xflag-FLI1 were transiently co-expressed with increasing concentrations (ratios 3xflag-protein to 3xmyc-USP19 1:2 and 1:4) of active 3xmyc-USP19 for 48 h in HEK293T cells. Lysates were analyzed by western blotting using anti-flag and anti-myc antibodies. Numbers below represent densitometrically quantified flag tagged protein over loading control GAPDH of a representative experiment.

Article Snippet: USP19 cDNA (Addgene #36306) was introduced into pCMV-3xflag or pCDNA-3xmyc vectors (tag N-terminal of USP19) by In-Fusion cloning HD (Clontech Laboratories Inc., Mountain View, CA, USA) according to manufacturer’s protocol.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Over Expression

Journal: Scientific Reports

Article Title: USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth

doi: 10.1038/s41598-018-37264-5

Figure Lengend Snippet: EWS-FLI1 ubiquitination is modulated by USP19. ( a ) EWS-FLI1 interacts with USP19. 3xflag-EWS-FLI1 and 3xmyc-USP19 were co-expressed in HEK293T cells for 48 h. After co-immunoprecipitation, lysates were analyzed by western blotting as indicated. ( b ) USP19 depletion increases EWS-FLI1 ubiquitination. A673 cells were transiently incubated with three different siRNAs against USP19 or a control siRNA for 24 h followed by co-expression of 3xflag-EWS-FLI1 and HA-ubiquitin for 48 h. Ubiquitination of EWS-FLI1 was analyzed by western blot analysis using anti-HA antibody. ( c ) EWSR1 also immunoprecipitates with USP19. 3xflag-EWS-FLI1, 3xflag-EWSR1 or 3xflag-FLI1 were co-expressed with 3xmyc-USP19 in HEK293 cells for 48 h. After co-immunoprecipitation, lysates were analyzed by western blotting as indicated. ( d ) USP19 depletion increases EWSR1 monoubiquitination. A673 cells were transiently incubated with two different siRNAs against USP19 or a control siRNA for 24 h followed by co-expression of 3xflag-EWSR1 and HA-ubiquitin for 48 h. Ubiquitination of EWSR1 was analyzed by western blot analysis using anti-HA antibody. Full-length blots of all immunoprecipitates are presented in Supplementary Fig. .

Article Snippet: USP19 cDNA (Addgene #36306) was introduced into pCMV-3xflag or pCDNA-3xmyc vectors (tag N-terminal of USP19) by In-Fusion cloning HD (Clontech Laboratories Inc., Mountain View, CA, USA) according to manufacturer’s protocol.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Incubation, Control, Expressing

Journal: Scientific Reports

Article Title: USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth

doi: 10.1038/s41598-018-37264-5

Figure Lengend Snippet: Depletion of USP19 affects Ewing sarcoma cell growth in vitro . ( a ) Scheme illustrating shRNA vector for stable transduction. The constitutively active part includes selection marker and a tetracycline repressive element. The doxycycline dependent part includes a tetracycline dependent promoter and the shRNA sequence. ( b ) SKNMC cells were stably transduced with two different shRNA sequences targeting USP19 and a control sequence (MOI = 5). After incubation with 0.1 ng/µl doxycycline for 72 h, USP19 protein levels were analyzed by western blotting using anti-USP19 antibody. Arrows indicate specific bands, asterisk marks unspecific band. ( c ) USP19 depletion affects cell growth. Knockdown of USP19 was induced in 2 × 10 4 SKNMC cells as indicated and cells were counted after 4 (smaller graph) and 8 days (larger graph). Total cell numbers were plotted from three independent experiments, error bars as SD. ( d ) Depletion of USP19 affects Ewing long-term cell survival. Doxycycline induced and non-induced SKNMC cells were plated to assess colony formation after 14 days. ( e ) Quantification of colonies from ( d ) represented as total counts of colony numbers (n = 3, error bars as SD). ( f , g ) Knockdown of USP19 affects cell proliferation and viability. Doxycycline induced and non-induced SKNMC cells were assessed for incorporation of BrdU or incubated with WST1 reagent ( g ) or both after 96 h. Values are shown relative to untreated shControl cells (n = 3, error bars as SD). ( h ,i) USP19 depletion has limited effect in unrelated non-tumorigenic cell lines. ( h ) MRC5 cells were transduced with two different shRNA sequences targeting USP19 and a control sequence and incubated with 0.1 ng/µl doxycycline for 72 h. USP19 protein levels were assessed by western blotting using anti-USP19 antibody, arrows indicate specific bands, asterisk marks unspecific band. (i) Doxycycline induced and non-induced MRC5 cells were assessed for cell viability by WST1 after 96 h (n = 3, error bars as SD).

Article Snippet: USP19 cDNA (Addgene #36306) was introduced into pCMV-3xflag or pCDNA-3xmyc vectors (tag N-terminal of USP19) by In-Fusion cloning HD (Clontech Laboratories Inc., Mountain View, CA, USA) according to manufacturer’s protocol.

Techniques: In Vitro, shRNA, Plasmid Preparation, Transduction, Selection, Marker, Sequencing, Stable Transfection, Control, Incubation, Western Blot, Knockdown

Journal: Scientific Reports

Article Title: USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth

doi: 10.1038/s41598-018-37264-5

Figure Lengend Snippet: Depletion of USP19 delays tumor growth in vivo . ( a ) Scheme of xenograft experiments using SKNMC inducible cell lines. ( b ) Doxycycline-induced shRNA knockdown against USP19 in vivo . Two mice with engrafted tumors of ~200 mm 3 (shCtr and shUSP19#1) were treated for five days with doxycycline. Tumor lysates were analyzed by western blotting with anti-USP19. ( c ) Tumor growth rate of indicated cell lines and treatment of subcutaneously injected SKNMC cells, error bars as SD. ( d ) Tumors of two representative mice transplanted with SKNMC shUSP19#1 cells and treated with doxycycline or PBS. ( e ) Immunohistochemical analysis of representative sections doxycycline or PBS treated SKNMC shUSP19#1 tumors using an USP19 antibody.

Article Snippet: USP19 cDNA (Addgene #36306) was introduced into pCMV-3xflag or pCDNA-3xmyc vectors (tag N-terminal of USP19) by In-Fusion cloning HD (Clontech Laboratories Inc., Mountain View, CA, USA) according to manufacturer’s protocol.

Techniques: In Vivo, shRNA, Knockdown, Western Blot, Injection, Immunohistochemical staining

Journal: Scientific Reports

Article Title: USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth

doi: 10.1038/s41598-018-37264-5

Figure Lengend Snippet: USP19 selectively stabilizes EWS-FLI1. ( a ) USP19 binds to the EWS-FLI1 fusion protein which is further deubiquitinated. ( b ) USP19 also binds to the stable EWSR1 full length protein resulting in removal of the monoubiquitin. As USP19 does not bind to full length FLI1, its polyubiquitination pattern is unaffected.

Article Snippet: USP19 cDNA (Addgene #36306) was introduced into pCMV-3xflag or pCDNA-3xmyc vectors (tag N-terminal of USP19) by In-Fusion cloning HD (Clontech Laboratories Inc., Mountain View, CA, USA) according to manufacturer’s protocol.

Techniques: