Journal: Communications Biology

Article Title: Plasmid2MC: efficient cell-free generation of high-purity minicircle DNA for genome editing in mammalian cells

doi: 10.1038/s42003-025-09157-7

Figure Lengend Snippet: a Diagram of a plasmid containing attP and attB recombination sites separating the bacterial backbone and the intended mcDNA. b Subunit rotation mechanism for strand exchange by serine recombinases (this subplot is adopted from Rutherford et al. 2013 ). c Random collision and topologically selective strand exchange both lead to intercoiled structures of two DNA loops of varying complexity among the recombined molecules . Random collision is the usual recombination mechanism, while topologically selective recombination was observed in experimental designs . d Gel image showing the ΦC31 recombination of 10418 bp plasmid into a 2.7 kbp bacterial backbone circle and 7758 bp DNA circle. Each lane contains 300 ng of DNA. Lane M - DNA ladder; L1 - pure supercoiled plasmid; L2 - same plasmid with a single cut with a restriction enzyme ( Not I); linear DNA travels faster than supercoiled DNA, and L1 presents a very weak matching band showing a minuscule amount of linear plasmid; L3 - after 80 minutes of recombination showing diffuse DNA scattering corresponding to the varying speed of travel of the miscellaneous catemers depicted in ( b ), and L4 same product cut with Not I, which is present in a single location on the bacterial backbone, thus allowing bacterial backbone release from the tangle, resulting in distinct bands (2.7 and 7.7 kbp) and a weaker band corresponding to the non-recombined plasmid; L7 and L8 - same as L3 and L4 after 12 hours of recombination; the original plasmid band is now nearly invisible; L9 and L10 - purified mcDNA following digestion of all non-circular DNA (see the unprocessed gel photos in Supplementary Fig. ).

Article Snippet: The plasmid p-att-ef1a-MS2-RecT-dCas9-BSD , 10482 bp in length, is a functional plasmid that was used as a basis for the recombination experiments (available at Addgene #226116).

Techniques: Plasmid Preparation, Purification

Journal: Communications Biology

Article Title: Plasmid2MC: efficient cell-free generation of high-purity minicircle DNA for genome editing in mammalian cells

doi: 10.1038/s42003-025-09157-7

Figure Lengend Snippet: a Temperature curve of ΦC31 recombination efficiency, showing the distinct maximum yield at 30 °C. b Recombination yield by time at the optimal temperature of 30 °C. c mcDNA yield at ΦC31 integrase concentrations of 25–200 ng/ μ l and a constant DNA concentration of 100 ng/ μ l. d mcDNA yield at 100 ng/ μ l ΦC31 and DNA concentrations of 100–300 ng/ μ l. e mcDNA yield at 200 ng/ μ l ΦC31 and DNA concentrations of 100–300 ng/ μ l. The following plots ( f – h ) account for the bacterial backbone DNA circle for complete recombination efficiency (including length of bacterial backbone, and discounting processing losses): f Recombination yield at ΦC31 concentrations of 25–200 ng/ μ l and a constant DNA concentration of 100 ng/ μ l. g Recombination yield at 100 ng/ μ l ΦC31 and DNA concentrations of 100–300 ng/ μ l. h Recombination yield at 200 ng/ μ l ΦC31 and DNA concentrations of 100–300 ng/ μ l. i Combining the data points shown above presented a molar ratio of ΦC31 to source plasmid DNA molecules. j Maximum yield test at ΦC31 concentration of 200 ng/ μ l and 20 μ g of input DNA at a concentration of 66.6 ng/ μ l. The column titled 1211/3871bp represents yields for an additional 1211 bp mcDNA produced out of 3871 bp parental plasmid. Note: Tests for each data point were performed in duplicate for all subplots, with exception of ( j ) performed in quadruplicate and triplicate, as seen in the subplot. Experiments in plots ( b – j ) were performed at 30 °C and 12 h incubation.

Article Snippet: The plasmid p-att-ef1a-MS2-RecT-dCas9-BSD , 10482 bp in length, is a functional plasmid that was used as a basis for the recombination experiments (available at Addgene #226116).

Techniques: Concentration Assay, Plasmid Preparation, Produced, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: Identification of USP19 as a TDP-43-directed deubiquitinase. ( A ) Schematic of Duolink® PLA principle for detection of ubiquitin (Ub)-TDP-43 complexes. ( B ) Representative images of Ub-TDP-43 PLA puncta (red), TDP-43 (green), and DAPI (blue) in tetracycline (tet)-induced HeLa-GFP-TDP-43 cells transfected with Smartpool siRNAs targeting 20 different USPs. ( C ) Quantification of Ub-TDP-43 PLA puncta numbers per cell (one-way ANOVA, F(20, 42) = 2.711, P = 0.0032; post hoc Dunnett compared to control siRNA (Cont), * P = 0.0270; n = 3 independent experiments per USP with 4 to 9 images averaged per experiment). ( D ) Schematic of USP19-flag constructs: ER-localized USP19 (USP19-ER), cytosolic USP19 (USP19-Cyto), and USP19-ER lacking catalytic DUB activity (USP19-CS-ER). ( E ) Representative blots showing co-IP of Myc-TDP-43 with USP19-flag variants (flag pulldown) in HEK293T cells cotransfected ± USP19-flag variants or vector control ± Myc-TDP-43. ( F ) Representative images of USP19–TDP-43 PLA puncta (red: myc + flag PLA) and DAPI (blue) from HeLa cells transfected with Myc-TDP-43 and USP19-flag variants or vector control. One probe - lacks one secondary antibody probe. ( G ) Representative blots of polyubiquitin conjugates (Ub-K48 & Ub-K63) in TDP-43 pulldown (myc) from HEK293T cells transfected with ubiquitin-HA (Ub-HA) and myc-TDP-43 with USP19-flag variants or vector control.

Article Snippet: The following plasmids were obtained from addgene: USP19-ER (#78597), USP19-Cyto (#78579), USP19-CS-ER (#78584), HA-Ubiquitin (#18712), USP19-ER-mCitrine (#78593), USP19-Cyto-mCitrine (#78595), USP19-CS-mCitrine (#78594), TDP-43-tomato-HA (#28205), and Cry2olig-mCherry (#60032). pcDNA (Invitrogen, Cat# V79020) was purchased from Invitrogen.

Techniques: Ubiquitin Proteomics, Transfection, Control, Construct, Activity Assay, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: USP19-ER isoform preferentially deubiquitinates and increases insoluble TDP-CTF through phase separation. ( A ) Representative blots of RIPA-soluble and RIPA-insoluble proteins from tet-induced GFP-TDP-43 cells transfected with vector control or USP19 variants. ( B – G ) Quantification of TDP-80 (GFP-TDP-43), TDP-43, and TDP-35 from RIPA-soluble (sol) and RIPA-insoluble (insol) fractions (Insol TDP-35: one-way ANOVA, F(3, 8) = 6.108, P = 0.0183; post hoc Dunnett, ** P < 0.01; ns: not significant; n = 3 independent experiments with 1 to 2 replicates averaged per experiment). ( H ) Representative blots of polyubiquitin conjugates (Ub-K48 & Ub-K63) in myc-TDP-35 pulldown (myc) from HEK293T cells transfected with ubiquitin-HA (Ub-HA) and myc-TDP-35 with USP19-flag variants or vector control. ( I ) Schematic model blue light-induced phase separation of Cry2olig-TDP-LCD-mCherry. ( J ) Representative images of Cry2olig-TDP-LCD-mCherry T(red) before and after blue light stimulation (488 nm, 1 s) in HeLa cells transfected with Cry2olig-TDP-LCD and USP19 variants (green) or vector control. ( K ) Quantification of Cry2olig-TDP-LCD puncta numbers normalized to cell area (two-way ANOVA, F(3, 39) = 21.33, P < 0.0001; post hoc Dunnett compared to control, **** P < 0.0001, ns: not significant; n = 2 independent experiments with 2 to 3 replicates averaged per experiment).

Article Snippet: The following plasmids were obtained from addgene: USP19-ER (#78597), USP19-Cyto (#78579), USP19-CS-ER (#78584), HA-Ubiquitin (#18712), USP19-ER-mCitrine (#78593), USP19-Cyto-mCitrine (#78595), USP19-CS-mCitrine (#78594), TDP-43-tomato-HA (#28205), and Cry2olig-mCherry (#60032). pcDNA (Invitrogen, Cat# V79020) was purchased from Invitrogen.

Techniques: Transfection, Plasmid Preparation, Control, Ubiquitin Proteomics

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:

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 promotes TBK1 degradation in a lysosome-dependent manner. (A) HEK293T cells were transfected with a MYC-TBK1 plasmid and increasing amounts of a Flag-USP19 expression plasmid and the extracted proteins were detected via immunoblotting. (B and C) HEK293T cells were transfected with MYC-TBK1 and Flag-USP19, then treated with CHX (100 μg/mL) for the indicated times after 24 h transfection. MYC-TBK1 protein levels were analyzed via immunoblotting (B). Myc-TBK1 expression was normalized to ACTB (C). (D and E) HEK293T cells were transfected with the indicated plasmids without (D) or with (E) a HA-Ub plasmid for 24 h, and then treated with MG132 (20 μM), CQ (50 μM), 3-MA (5 mM) or Baf-A1 (20 nM) for 6 h. The proteins were then detected via immunoblotting. (F) HEK293T cells were transfected with MYC-TBK1, HA-Ub, and Flag-USP19 plasmids for 24 h, then the cells were treated with MG132 (20 μM) and CQ (50 μM) for 6 h before harvest. The ubiquitination experiment was performed and the results were analyzed via immunoblotting. (G) HEK293T cells were transfected with MYC-TBK1, Flag-USP19, or Flag-USP19C607S plasmids, and the extracted proteins were detected via immunoblotting. The data are representative of three independent experiments. Error bars show the means ± SD. *p < 0.05, **p < 0.01 using the student’s t test.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 interacts with TBK1. (A) HEK293T cells were transfected with MYC-TBK1 and Flag-USP19 plasmids, and then subjected to immunoprecipitation with a Flag antibody before immunoblot analysis. (B and C) HeLa (B) or THP1 cells (C) were infected with VSV for the indicated times, and then subjected to immunoprecipitation with a TBK1 antibody before immunoblot analysis of endogenous TBK1and USP19 protein levels. (D) HEK293T cells were transfected with MYC-TBK1 or Flag-USP19 plasmids, using immunoprecipitation to purify the proteins using Flag or MYC beads. The purified proteins, including Flag-usp19 and MYC-TBK1, were mixed in a Co-IP buffer to perform an in vitro pull-down assay and the results were analyzed via Coomassie Brilliant Blue staining and an immunoblot assay. (E) HEK293T cells were transfected with MYC-TBK1, Flag-USP19, or Flag-USP19C607S plasmids, and then subjected to immunoprecipitation with a Flag antibody before immunoblot analysis. (F) Schematic of TBK1 and the derivatives used (top). HEK293T cells were transfected with MYC-TBK1 (WT), MYC-TBK1 (KD), MYC-TBK1 (KD+ULD), MYC-TBK1 (ULD+CC), and Flag-USP19 plasmids, and then analyzed via immunoprecipitation and immunoblotting with the indicated antibodies (bottom). (G) Schematic of USP19 and the derivatives used (top). HEK293T cells were transfected with Flag-USP19 (WT), Flag-USP19 (CS), Flag-USP19 (USP), Flag-USP19 (USP+TMD), and MYC-TBK1, and then analyzed via immunoprecipitation and immunoblotting with the indicated antibodies (bottom). The data are representative of three independent experiments.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Purification, Co-Immunoprecipitation Assay, In Vitro, Pull Down Assay, Staining

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 promotes TBK1 degradation through the CMA-dependent autophagy pathway. (A) HeLa cells were transfected with Flag-USP19 or Flag-USP19C607S plasmid and the LC3B levels were analyzed via immunoblotting. (B) Alignment of TBK1 orthologs. The pink shading indicates the conserved CMA motif. (C) HEK293T cells were transfected with MYC-TBK1 and Flag-USP19 plasmids for 24 h, labeled with the indicated antibodies, and analyzed via confocal microscopy. (D) HEK293T cells were transfected with MYC-TBK1, Flag-USP19, V5-LAMP2A, and HA-HSPA8 plasmids for 24 h, and then the lysates were immunoprecipitated with an anti-Flag antibody and analyzed via immunoblotting. (E) HeLa cells were infected with or without VSV, then the lysosomes were isolated and enriched before the proteins were analyzed via immunoblotting. (F) HeLa cells were transfected with MYC-TBK1, Flag-USP19, V5-LAMP2A, and HA-HSPA8 plasmids before analysis via immunoblotting with the indicated antibodies. (G) HeLa cells were transfected with MYC-TBK1, Flag-USP19, and V5-LAMP2A plasmids, and then treated with CQ (50 μM) before the proteins were analyzed via immunoblotting. (H) Tbk1-knockout Raw 264.7 cells were transfected with Flag-USP19, MYC-TBK1, or MYC-TBK1Q588A plasmids, and the indicated proteins were analyzed via immunoblotting. (I and J) HeLa cells were transfected with increasing amounts of Flag-USP19 plasmids for 24 h before qPCR analysis of LAMP2A (I) and HSPA8 (J) mRNA expression. (K) HeLa cells were transfected with increasing amounts of Flag-USP19 plasmids for 24 h before analysis of LAMP2A and HSPA8 protein levels via immunoblotting. (L) HeLa cells were transfected with increasing amounts of Flag-USP19 plasmids for 24 h, subjected to immunoprecipitation with an anti-HSPA8 antibody, and the results were analyzed via immunoblotting. (M and N) HeLa cells were transfected with gene-specific siRNA to knock down LAMP2A (M) or HSPA8 (N) mRNA expression, and were then transfected with MYC-TBK1 and Flag-USP19 plasmids. MYC-TBK1 protein levels were analyzed via immunoblotting. (O and P) HEK293T cells were transfected with a Flag-USP19 plasmid, with (O) or without (P) MYC-TBK1 plasmids, and then treated with VER-155,008 (5 μM) or Torin 1 (250 nM). MYC-TBK1 or endogenous TBK1 protein levels were analyzed via immunoblotting. Scale bars: 5 μm. The data are representative of three independent experiments. Error bars show the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Transfection, Plasmid Preparation, Western Blot, Labeling, Confocal Microscopy, Immunoprecipitation, Infection, Isolation, Knock-Out, Expressing

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 negatively regulates VSV-induced IFNB production and the antiviral response in THP1 cells. (A-G) THP1 cells were transfected with control or USP19 siRNA for 48 h, with or without pretreatment with VER-155,008 (5 μM) for 30 min, then USP19 protein levels were analyzed via immunoblotting (A). qPCR analysis of IFNB1 expression for the indicated times after VSV infection (MOI = 1) (B). ELISA analysis of IFNB production in the cell culture supernatants 12 h after VSV infection (C). qPCR analysis of CCL5 and CXCL10 expression 12 h after VSV infection (MOI = 1) (D). qPCR analysis of ISG15, IFIT2, IFIT1, and MX1 expression 12 h after VSV infection (MOI = 1) (E). THP-1 cells were infected with VSV-GFP (MOI = 1) for 12 h and the cells were imaged under a confocal microscope (F). The percentage of GFP+ cells was determined via flow cytometry (G). (H) THP1 cells were transfected with control or USP19 siRNA, and then infected with VSV. Immunoblotting was performed to analyze TBK1 protein levels. (I-M) THP1 cells were transfected with Flag-USP19 plasmids or an empty vector for 24 h, with or without pretreatment with VER-155,008 (5 μM) for 30 min. USP19 expression was analyzed via immunoblotting using a Flag antibody (I). qPCR analysis of IFNB1 (J), CCL5, CXCL10 (K), ISG15, IFIT2, IFIT1, and MX1 (L) expression and VSV replication (M) for the indicated times after VSV infection (MOI = 1). (N) THP1 cells were transfected with Flag-USP19 plasmids or an empty vector, and then infected with VSV. Immunoblotting was performed to analyze TBK1 protein levels. (O) THP1 cells were transfected with Flag-USP19C607S plasmids or an empty vector, and then infected with VSV. Immunoblotting was performed to analyze indicated proteins levels. The data are representative of three independent experiments. Scale bars: 100 μm. Error bars show the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Transfection, Western Blot, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture, Microscopy, Flow Cytometry, Plasmid Preparation

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 negatively regulates antiviral signaling by targeting TBK1. (A-F) HEK293T cells were transfected with different concentrations of USP19 plasmids and DDX58 (A and B), MAVS (C and D) or TBK1 (E and F) plasmids. Dual-luciferase reporter assays were performed 24 h after transfection. Exogenous USP19 protein levels were analyzed via immunoblotting. (G) HEK293T cells were transfected with MYC-DDX58, MYC-MAVS, MYC-TBK1, MYC-IRF3, MYC-IFIH1, His-CGAS, His-STING1, His-TBK1, and Flag-USP19 expression plasmids for 24 h before the lysates were subjected to immunoprecipitation with an anti-MYC or His antibody, and the results were analyzed via immunoblotting. The data are representative of three independent experiments. Error bars show the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Transfection, Luciferase, Western Blot, Expressing, Immunoprecipitation

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: Knockdown of USP19 promotes cellular antiviral response in human PBMCs. (A-E) PBMCs were transfected with control or USP19 siRNAs for 48 h, with or without pretreatment with VER-155,008 (5 μM) for 30 min. USP19 expression was analyzed via qPCR (A) and immunoblotting (B). qPCR analysis of IFNB1 expression 12 h after VSV infection (MOI = 1) (C). ELISA analysis of IFNB production in PBMC supernatants 12 h after VSV infection (MOI = 1) (D). qPCR analysis of VSV replication 12 h after VSV infection (MOI = 1) (E). (F and G) PBMCs were transfected with control or USP19 siRNAs for 48 h, VSV infection (MOI = 1) was performed at the indicated times, and the indicated proteins were analyzed via immunoblotting. The data are representative of three independent experiments. Error bars show the means ± SD. *p < 0.05, **p < 0.01 using Student’s t test.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Transfection, Expressing, Western Blot, Infection, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 deficiency in macrophages enhanced VSV-induced type I interferon production and antiviral response by suppressing CMA-mediated TBK1 degradation. (A and B) qPCR analysis of Ifna4 (A) and Ifnb1 (B) expression in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated times with or without VER-155,008 (5 μM) pretreatment. (C and D) qPCR analysis of Ifna4 (C) and Ifnb1 (D) expression in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages transfected with poly(I:C) (1 μg/mL) for the indicated hours with or without pretreatment with VER-155,008 (5 μM). (E and F) qPCR analysis of Ccl5, Cxcl10 (E), Isg15, Ifit2, Ifit1, and Mx1 expression (F) in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for 12 h with or without pretreatment with VER-155,008 (5 μM). (G) Immunoblot analysis of OSA1, EIF2AK2, and LAMP2A expression in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated durations. (H and I) Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages were infected with VSV-GFP for 12 h with or without VER-155,008 (5 μM) pretreatment, and images were captured under a fluorescence microscope (H). The percentage of GFP+ cells was determined via flow cytometry (I). (J) qPCR analysis of VSV replication in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) with or without VER-155,008 (5 μM) pretreatment. (K) Determination of VSV titers in supernatants via a TCID50 assay of Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV with or without VER-155,008 (5 μM) treatment. (L) Immunoblot analysis of the indicated proteins in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated durations. (M) Immunoblot analysis of STAT1 and STAT2 phosphorylation in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated hours. The data are representative of three independent experiments. Scale bars: 100 μm. Error bars show the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Expressing, Infection, Transfection, Western Blot, Fluorescence, Microscopy, Flow Cytometry, TCID50 Assay

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 macrophage-deficient mice show resistance to VSV infection. (A and B) qPCR analysis of Ifna4 (A) and Ifnb1 (B) mRNA expression in organs from Usp19flox/flox and usp19flox/flox Lyz2-Cre mice 12 h after VSV administration (1 × 108 PFU/g) via intraperitoneal injection (n = 3 per group) with or without VER-155,008 (40 mg/kg) treatment. (C) Determination of VSV loads in organs through the TCID50 assay using Usp19flox/flox and usp19flox/flox Lyz2-Cre mice (n = 3 per group). (D) ELISA analysis of IFNB production in sera from Usp19flox/flox and usp19flox/flox Lyz2-Cre mice (n = 3 per group). Error bars show means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test. (E) Pathology of Usp19flox/flox and usp19flox/flox Lyz2-Cre mice in response to VSV infection with or without pretreatment with VER-155,008 (40 mg/kg). Scale bar: 200 μm. (F) Survival of Usp19flox/flox and usp19flox/flox Lyz2-Cre mice administered with VSV (1 × 108 PFU/g) via intraperitoneal injection (n = 8–10 per group). Statistical significance was calculated using the Log-rank (Mantel-Cox) test. The data are representative of three independent experiments.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: Infection, Expressing, Injection, TCID50 Assay, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: Schematic representation of the role of USP19 in the regulation of TBK1 degradation via CMA. Pattern recognition receptors (PRRs, e.g., RLRs, TLRs, and CGAS) can recognize pathogen-associated molecular patterns (PAMPs, e.g., ssRNA, dsRNA, dsDNA, and LPS) and recruit different receptor proteins (e.g., MAVS, STING1, and TICAM1/TRIF) to activate TBK1. USP19 interacts with the TBK1-HSPA8 complex and then promotes TBK1 degradation via CMA, negatively regulating type I interferon production.

Article Snippet: Next, purified Flag-USP19 and MYC-TBK1 proteins were mixed in the NP-40 lysis buffer (Beyotime Biotechnology, P0013 F) for co-immunoprecipitation and analyzed via an immunoblot assay.

Techniques: