Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ER-anchored USP19 promotes the secretion of misfolded TDP-43 in the HEK293T cellular model. a Schematic representation of Flag-USP19 constructs used in this study. The C506 residue represents an essential amino acid residue necessary for the ubiquitin peptidase catalytic activity. CS 1&2 CHORD-containing proteins and STG1, UBL ubiquitin-like, USP ubiquitin-specific peptidase, TM transmembrane domain, ZnF zinc finger, ΔTM deleted for the transmembrane domain. The pink star represents the amino terminal Flag tag. b Schematic representation of the full-length TDP-43 (upper panel). The K263E variant is highlighted in blue. LCD Low Complexity Domain, NLS nuclear localization sequences, NTD N-terminal domain, RRM1&2 RNA recognition motif 1&2. HEK293T cells were transfected with TDP-43-WT or TDP-43-K263E encoding constructs and were visualized by immunofluorescence using anti-TDP-43 antibody. Blue signal corresponds to DAPI for nuclei and green signal to TDP-43. Scale bar is 10 µm. c Immunoblotting of sarkosyl soluble supernatant (Sark-sol) and sarkosyl insoluble pellet (Sark-ins) fractions isolated from control HEK293T cells (lanes 1 and 4), or cells expressing TDP-43-WT (lanes 2 and 5) or TDP-43-K263E (lanes 3 and 6) using antibodies directed against TDP-43 and GAPDH as loading control. d Evaluation of misfolded TDP-43 secretion by filter trap assay (FTA). Presence of misfolded TDP-43 in conditioned media from HEK293T cells overexpressing TDP-43-WT and TDP-43-K263E and the different Flag-USP19 or the empty vector (negative control) was monitored by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using anti-Flag (for USP19), -TDP-43 and -GAPDH antibodies for loading control. e Quantification of secreted TDP-43 upon USP19 expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test ( **p < 0.001 ). f USP19-WT or USP19-ΔTM and TDP-43-K263E overexpression does not affect plasma membrane permeability and cell viability. HEK293T cells transfected with the indicated plasmids were stained with trypan blue and counted. Data represent mean ± SEM, n = 3 experiments

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Construct, Residue, Ubiquitin Proteomics, Activity Assay, FLAG-tag, Variant Assay, Transfection, Immunofluorescence, Western Blot, Isolation, Control, Expressing, TRAP Assay, Plasmid Preparation, Negative Control, MANN-WHITNEY, Over Expression, Clinical Proteomics, Membrane, Permeability, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ubiquitin peptidase activity is essential for TDP-43-K263E secretion in USP19 context. a Schematic representation of Flag-USP19 constructs. The USP19-C506S corresponds to an ER-anchored but ubiquitin peptidase catalytically inactive mutant. b Ubiquitination level in cell lysates of co-expressing cells. Immunoblotting of cell lysates from TDP-43-K263E and the indicated Flag-USP19 co-expressing cell using antibodies directed against Ubiquitin or GAPDH as loading control. c Evaluation of misfolded TDP-43 secretion by FTA. Presence of misfolded TDP-43 in conditioned media from HEK293T cells overexpressing the TDP-43-K263E, the WT, the ΔTM or the C506S Flag-USP19, was monitored by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using anti-Flag (for USP19), -TDP-43 and -GAPDH antibodies for loading control. d Quantification of secreted TDP-43-K263E upon USP19s expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test (** p < 0.001). e TDP-43-K263E deubiquitination is promoted by USP19-WT. Lysates from HEK293T cells co-expressing Myc-TDP-43-K263E and HA-Ubiquitin-WT with Flag-USP19-C506S (ubiquitin peptidase deficient mutant; lane 1) or Flag-USP19-WT (lane 2) were immunoprecipitated with anti-Myc and immunoblotted with anti-HA and anti-Myc. Whole cell lysates (WCL; 5 μg/input) from co-expressing cells were immunoblotted using anti-Myc, anti-Flag and anti-GAPDH for loading control. Asterisks correspond to heavy and light chains of immunoglobulins used during the immunoprecipitation

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Ubiquitin Proteomics, Activity Assay, Construct, Mutagenesis, Expressing, Western Blot, Control, MANN-WHITNEY, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ER-anchored USP19 promotes the secretion of free TDP-43-K263E fibrils in the conditioned medium. a Transmission of misfolded HA-TDP-43-K263E to naïve HEK293T cells. Naive HEK293T cells were cocultured with conditioned media from HEK293T co-expressing cells Flag-USP19-WT or − ΔTM with HA-TDP-43-K263E during 72 h. b After 3 days, cells were washed and analyzed by Western blotting using anti-HA and anti-GAPDH as loading control. c Quantification of n = 5 independent experiments. Significance was assessed by a Mann–Whitney U test (**p < 0.001). d Aggregated TDP-43 levels in conditioned media from HEK293T cells overexpressing TDP-43-K263E and the Flag-USP19 WT and ΔTM were precleared to eliminate cellular debris and ultracentrifuged at 120,000 g pellet (p120K pellet) and analyzed by immunoblotting (lanes 3 and 4) using antibody directed against TDP-43. Cellular lysates (lanes 1 and 2) were analyzed by Western blotting and probed by antibodies directed against TDP-43, Flag and GAPDH (loading control). e Sucrose equilibrium density gradient fractionation of conditioned medium. The p120K pellet was fractionated through a linear sucrose equilibrium density gradient 9–60% and fractions (× 15), recovered from the top of the gradient, were analyzed by Western blotting using anti-TDP-43 or anti-CD81 and anti-CD63 antibodies for extracellular vesicles markers. Fraction density values (g/cm 3 ) are depicted at the bottom panel. Whole cell lysate (WCL) of co-expressing cells was immunoblotted in parallel using the anti-TDP-43, anti-CD63 and anti-CD81. The bottom panel corresponds to the TCE staining of the SDS-PAGE gel. f Immunogold electron microscopy (IEM) of TDP-43-K263E positive fractions. Positive fractions (10–12) containing the TDP-43-K263E were pooled and analyzed by IEM using anti-TDP-43 labelled with a secondary antibody coupled with 10 nm gold particle. Amorphous and fibrillar structures were labelled (red arrows). Scale bars are 20 and 50 nm

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Transmission Assay, Expressing, Western Blot, Control, MANN-WHITNEY, Fractionation, Staining, SDS Page, Electron Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: Transmission electron microscopy (TEM) and RNA sequencing analyses point out ER alterations in TDP-43-K263E and USP19-WT co-expressing cells. a Ultrastructural analyses. TEM of TDP-43-K263E + UPS19-WT (panel i); TDP-43-K263E (panel ii); TDP-43-K263E + USP19-ΔTM (panel iii) and untransfected cells (UT; panel iv as negative control). Black arrows indicate dilated endoplasmic reticulum (ER) accumulation (panels i and v; v-right corresponds to a higher magnification of panel v). White asterisk indicates cytoplasmic aggregates (panel ii). White arrows indicate compact electron dense structures (CEDS; panel iii). Red arrows indicate mitochondria in close contacts with ER (panels v-right to vii). Yellow arrowheads indicate autophagic/endosomal compartments containing ER membrane and dense amorphous structures. (panels v-right, x and xi). Scale bars are 2 and 5 μm. Dotted squares correspond to higher magnification b Volcano plot of differentially expressed genes between TDP-43-K263E/USP19-WT versus TDP-43-K263E/USP19-ΔTM co-expressing cells. Red dots represents upregulated genes (P < 0.05 and Log2FC > 1), blue dots represents downregulated genes (P < 0.05 and Log2FC < -1), and grey dots represent genes that were not differentially expressed. c Bubble plot of the GO Biological process pathway enrichment analysis. The horizontal axis represents the enrichment score (-log10(p-value)), while the vertical axis represents the enriched pathway name. The color scale indicates different thresholds of the p-value, and size of the bubble indicates the number of genes corresponding to each pathway

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Transmission Assay, Electron Microscopy, RNA Sequencing, Expressing, Negative Control, Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: TDP-43-K263E colocalizes with USP19-WT and the KDEL-ER marker and coimmunoprecipitates with USP19-WT. a TDP-43 IEM of HEK293T TDP-43-K263E + Flag-WT-USP19 co-expressing cells. Red arrows indicate the TDP-43 gold particles in close contacts with dilated ER structures in the cytoplasm (panel i) or inside autophagic/endosomal compartments (panels iii and iv). Red arrowheads correspond to free TDP-43 aggregates in intracellular compartments (panel ii). White arrowheads correspond to TDP-43 labelling embedded in amorphous structures present in intracellular autophagic/endosomal compartments (panel iv). b Confocal immunofluorescence imaging of TDP-43-K263E + Flag-USP19-WT co-expressing cells. Co-expressing cells were labelled with antibodies directed against the ER KDEL marker (red), the Flag-USP19-WT (magenta), the HA for TDP-43-K263E (green) and the DAPI (blue) for nuclear staining. Scale bar is 10 μm. c ROI 1–2 are depicted in the merge panel. The plot profiles of the ER-KDEL marker (red) with the Flag-USP19-WT (magenta) and the HA-TDP-43-K263E (green) colocalizations (ROI1 and 2) along the ROI lines were constructed and analyzed using Image J software. White arrowheads show other colocalized signals d TDP-43 coimmunoprecipitates with Flag-USP19-WT. Left panel: co-immunoprecipitation experiment was realized on cell lysates from TDP-43-K263E and Flag-USP19-WT co-expressing cells using mouse antibodies directed against the Flag epitope or an irrelevant SARS-CoV2 IgG antibody as negative control. Right panel: Western blotting of HA-TDP-43-K263E and Flag-USP19-WT co-expressing cell lysates (input) using antibodies directed against Flag (for USP19), HA (for TDP-43) and GAPDH

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Marker, Expressing, Immunofluorescence, Imaging, Staining, Construct, Software, Immunoprecipitation, FLAG-tag, Negative Control, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: Early autophagic and endosomal compartments are involved in the TDP-43-K263E secretion mediated by USP19-WT. a Schematic representation of cellular trafficking and inhibition strategies (pharmacological in blue and siRNAs in red) used in this study to identify potential pathways involved in the TDP-43-K263E secretion mediated by USP19. b-f Evaluation of the TDP-43-K263E secretion mediated by USP19 by FTA in presence of siRNAs control (CT) or siRNAs directed against ATG7 , RAB11A , HRS/HGS , RAB8A or RAB27A . Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from siRNAs CT/targets-treated co-expressing cells using antibodies directed against ATG7, RAB11A, HRS/HGS, RAB8, RAB27A or Flag (for USP19), TDP-43, and GAPDH for loading control. Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of siRNAs CT and targets ATG7 , RAB11A , HRS/HGS , RAB8A and RAB27A are depicted in the right panels of each condition. Data represent mean ± SEM, n = 4 to 6 experiments. Significance was assessed by a Mann–Whitney U test (*p < 0.05)

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Inhibition, Control, Expressing, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: VAMP7 is a modulator of the TDP-43-K263E secretion mediated by USP19-WT. a Evaluation of the TDP-43-K263E secretion mediated by EGFP-VAMP7-WT and Flag-USP19-WT by FTA. Upper panel (secretion): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for USP19), TDP-43, GFP (for VAMP7-WT) and GAPDH for loading control. b Quantification of secreted TDP-43-K263E in presence of GFP-VAMP7-WT or Flag-USP19-WT expression. Data represent mean ± SEM, n = 4 experiments. Significance was assessed by a Mann–Whitney U test, ns = not significant. c Sucrose equilibrium density gradient fractionation of conditioned medium. The 120,000xg pellet (p120K) from conditioned media of TDP-43-K263E + GFP-VAMP7-WT co-expressing cells were fractionated through a 8–60% linear sucrose equilibrium density gradient and fractions (× 15), recovered from the top were analyzed by Western blotting using anti-TDP-43 or anti-CD81 antibody. Fraction density values (g/cm 3 ) are depicted at the bottom panel. d Evaluation of TDP-43-K263E secretion mediated by USP19-WT in presence of EGFP- VAMP7-WT, EGFP-Longin-VAMP7, EGFP-YKT6-WT or EGFP-Longin-YKT6 by FTA. Upper panel (secretion/conditioned medium): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for USP19), TDP-43, GFP (for VAMP7s and YKT6s) and GAPDH for loading control. e Quantification of secreted TDP-43-K263E mediated by Flag- USP19-WT in presence of VAMP7 or YKT6-WT and -Longins expression. Data represent mean ± SEM, n = 4 experiments. Significance was assessed by a Mann–Whitney U test (*p < 0.05), ns = not significant

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY, Fractionation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: DNAJC5/CSPα is a modulator of the misfolded TDP-43-K263E secretion mediated by Flag-USP19-WT. a Evaluation of TDP-43-K263E secretion mediated by 3x-Flag-CSPα-WT or the phospho-deficient CSPα-S10A by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against HA (for HA tagged TDP-43), CSPα and GAPDH for loading control. b Evaluation of the TDP-43-K263E secretion mediated by USP19-WT in presence of CSPα-WT or CSPα-S10A mutant by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for Flag-USP19-WT), TDP-43, CSPα and GAPDH for loading control. c Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of CSPα-WT or the phosphor-deficient CSPα-S10A expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test (**p < 0.001). d Evaluation of TDP-43-K263E secretion mediated by Flag-USP19-WT in presence of siRNAs CT or siRNAs directed against CSPα by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells treated with siRNAs CT or against CSPα using antibodies directed against CSPα, Flag (for Flag-USP19-WT), TDP-43, or GAPDH for loading control. e Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of siRNAs CT or siRNAs CSPα. Data represent mean ± SEM, n = 3 experiments

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Expressing, Western Blot, Control, Mutagenesis, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ER-anchored USP19 promotes the secretion of misfolded TDP-43 in the HEK293T cellular model. a Schematic representation of Flag-USP19 constructs used in this study. The C506 residue represents an essential amino acid residue necessary for the ubiquitin peptidase catalytic activity. CS 1&2 CHORD-containing proteins and STG1, UBL ubiquitin-like, USP ubiquitin-specific peptidase, TM transmembrane domain, ZnF zinc finger, ΔTM deleted for the transmembrane domain. The pink star represents the amino terminal Flag tag. b Schematic representation of the full-length TDP-43 (upper panel). The K263E variant is highlighted in blue. LCD Low Complexity Domain, NLS nuclear localization sequences, NTD N-terminal domain, RRM1&2 RNA recognition motif 1&2. HEK293T cells were transfected with TDP-43-WT or TDP-43-K263E encoding constructs and were visualized by immunofluorescence using anti-TDP-43 antibody. Blue signal corresponds to DAPI for nuclei and green signal to TDP-43. Scale bar is 10 µm. c Immunoblotting of sarkosyl soluble supernatant (Sark-sol) and sarkosyl insoluble pellet (Sark-ins) fractions isolated from control HEK293T cells (lanes 1 and 4), or cells expressing TDP-43-WT (lanes 2 and 5) or TDP-43-K263E (lanes 3 and 6) using antibodies directed against TDP-43 and GAPDH as loading control. d Evaluation of misfolded TDP-43 secretion by filter trap assay (FTA). Presence of misfolded TDP-43 in conditioned media from HEK293T cells overexpressing TDP-43-WT and TDP-43-K263E and the different Flag-USP19 or the empty vector (negative control) was monitored by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using anti-Flag (for USP19), -TDP-43 and -GAPDH antibodies for loading control. e Quantification of secreted TDP-43 upon USP19 expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test ( **p < 0.001 ). f USP19-WT or USP19-ΔTM and TDP-43-K263E overexpression does not affect plasma membrane permeability and cell viability. HEK293T cells transfected with the indicated plasmids were stained with trypan blue and counted. Data represent mean ± SEM, n = 3 experiments

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Construct, Residue, Ubiquitin Proteomics, Activity Assay, FLAG-tag, Variant Assay, Transfection, Immunofluorescence, Western Blot, Isolation, Control, Expressing, TRAP Assay, Plasmid Preparation, Negative Control, MANN-WHITNEY, Over Expression, Clinical Proteomics, Membrane, Permeability, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ubiquitin peptidase activity is essential for TDP-43-K263E secretion in USP19 context. a Schematic representation of Flag-USP19 constructs. The USP19-C506S corresponds to an ER-anchored but ubiquitin peptidase catalytically inactive mutant. b Ubiquitination level in cell lysates of co-expressing cells. Immunoblotting of cell lysates from TDP-43-K263E and the indicated Flag-USP19 co-expressing cell using antibodies directed against Ubiquitin or GAPDH as loading control. c Evaluation of misfolded TDP-43 secretion by FTA. Presence of misfolded TDP-43 in conditioned media from HEK293T cells overexpressing the TDP-43-K263E, the WT, the ΔTM or the C506S Flag-USP19, was monitored by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using anti-Flag (for USP19), -TDP-43 and -GAPDH antibodies for loading control. d Quantification of secreted TDP-43-K263E upon USP19s expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test (** p < 0.001). e TDP-43-K263E deubiquitination is promoted by USP19-WT. Lysates from HEK293T cells co-expressing Myc-TDP-43-K263E and HA-Ubiquitin-WT with Flag-USP19-C506S (ubiquitin peptidase deficient mutant; lane 1) or Flag-USP19-WT (lane 2) were immunoprecipitated with anti-Myc and immunoblotted with anti-HA and anti-Myc. Whole cell lysates (WCL; 5 μg/input) from co-expressing cells were immunoblotted using anti-Myc, anti-Flag and anti-GAPDH for loading control. Asterisks correspond to heavy and light chains of immunoglobulins used during the immunoprecipitation

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Ubiquitin Proteomics, Activity Assay, Construct, Mutagenesis, Expressing, Western Blot, Control, MANN-WHITNEY, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ER-anchored USP19 promotes the secretion of free TDP-43-K263E fibrils in the conditioned medium. a Transmission of misfolded HA-TDP-43-K263E to naïve HEK293T cells. Naive HEK293T cells were cocultured with conditioned media from HEK293T co-expressing cells Flag-USP19-WT or − ΔTM with HA-TDP-43-K263E during 72 h. b After 3 days, cells were washed and analyzed by Western blotting using anti-HA and anti-GAPDH as loading control. c Quantification of n = 5 independent experiments. Significance was assessed by a Mann–Whitney U test (**p < 0.001). d Aggregated TDP-43 levels in conditioned media from HEK293T cells overexpressing TDP-43-K263E and the Flag-USP19 WT and ΔTM were precleared to eliminate cellular debris and ultracentrifuged at 120,000 g pellet (p120K pellet) and analyzed by immunoblotting (lanes 3 and 4) using antibody directed against TDP-43. Cellular lysates (lanes 1 and 2) were analyzed by Western blotting and probed by antibodies directed against TDP-43, Flag and GAPDH (loading control). e Sucrose equilibrium density gradient fractionation of conditioned medium. The p120K pellet was fractionated through a linear sucrose equilibrium density gradient 9–60% and fractions (× 15), recovered from the top of the gradient, were analyzed by Western blotting using anti-TDP-43 or anti-CD81 and anti-CD63 antibodies for extracellular vesicles markers. Fraction density values (g/cm 3 ) are depicted at the bottom panel. Whole cell lysate (WCL) of co-expressing cells was immunoblotted in parallel using the anti-TDP-43, anti-CD63 and anti-CD81. The bottom panel corresponds to the TCE staining of the SDS-PAGE gel. f Immunogold electron microscopy (IEM) of TDP-43-K263E positive fractions. Positive fractions (10–12) containing the TDP-43-K263E were pooled and analyzed by IEM using anti-TDP-43 labelled with a secondary antibody coupled with 10 nm gold particle. Amorphous and fibrillar structures were labelled (red arrows). Scale bars are 20 and 50 nm

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Transmission Assay, Expressing, Western Blot, Control, MANN-WHITNEY, Fractionation, Staining, SDS Page, Electron Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: Transmission electron microscopy (TEM) and RNA sequencing analyses point out ER alterations in TDP-43-K263E and USP19-WT co-expressing cells. a Ultrastructural analyses. TEM of TDP-43-K263E + UPS19-WT (panel i); TDP-43-K263E (panel ii); TDP-43-K263E + USP19-ΔTM (panel iii) and untransfected cells (UT; panel iv as negative control). Black arrows indicate dilated endoplasmic reticulum (ER) accumulation (panels i and v; v-right corresponds to a higher magnification of panel v). White asterisk indicates cytoplasmic aggregates (panel ii). White arrows indicate compact electron dense structures (CEDS; panel iii). Red arrows indicate mitochondria in close contacts with ER (panels v-right to vii). Yellow arrowheads indicate autophagic/endosomal compartments containing ER membrane and dense amorphous structures. (panels v-right, x and xi). Scale bars are 2 and 5 μm. Dotted squares correspond to higher magnification b Volcano plot of differentially expressed genes between TDP-43-K263E/USP19-WT versus TDP-43-K263E/USP19-ΔTM co-expressing cells. Red dots represents upregulated genes (P < 0.05 and Log2FC > 1), blue dots represents downregulated genes (P < 0.05 and Log2FC < -1), and grey dots represent genes that were not differentially expressed. c Bubble plot of the GO Biological process pathway enrichment analysis. The horizontal axis represents the enrichment score (-log10(p-value)), while the vertical axis represents the enriched pathway name. The color scale indicates different thresholds of the p-value, and size of the bubble indicates the number of genes corresponding to each pathway

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Transmission Assay, Electron Microscopy, RNA Sequencing, Expressing, Negative Control, Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: TDP-43-K263E colocalizes with USP19-WT and the KDEL-ER marker and coimmunoprecipitates with USP19-WT. a TDP-43 IEM of HEK293T TDP-43-K263E + Flag-WT-USP19 co-expressing cells. Red arrows indicate the TDP-43 gold particles in close contacts with dilated ER structures in the cytoplasm (panel i) or inside autophagic/endosomal compartments (panels iii and iv). Red arrowheads correspond to free TDP-43 aggregates in intracellular compartments (panel ii). White arrowheads correspond to TDP-43 labelling embedded in amorphous structures present in intracellular autophagic/endosomal compartments (panel iv). b Confocal immunofluorescence imaging of TDP-43-K263E + Flag-USP19-WT co-expressing cells. Co-expressing cells were labelled with antibodies directed against the ER KDEL marker (red), the Flag-USP19-WT (magenta), the HA for TDP-43-K263E (green) and the DAPI (blue) for nuclear staining. Scale bar is 10 μm. c ROI 1–2 are depicted in the merge panel. The plot profiles of the ER-KDEL marker (red) with the Flag-USP19-WT (magenta) and the HA-TDP-43-K263E (green) colocalizations (ROI1 and 2) along the ROI lines were constructed and analyzed using Image J software. White arrowheads show other colocalized signals d TDP-43 coimmunoprecipitates with Flag-USP19-WT. Left panel: co-immunoprecipitation experiment was realized on cell lysates from TDP-43-K263E and Flag-USP19-WT co-expressing cells using mouse antibodies directed against the Flag epitope or an irrelevant SARS-CoV2 IgG antibody as negative control. Right panel: Western blotting of HA-TDP-43-K263E and Flag-USP19-WT co-expressing cell lysates (input) using antibodies directed against Flag (for USP19), HA (for TDP-43) and GAPDH

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Marker, Expressing, Immunofluorescence, Imaging, Staining, Construct, Software, Immunoprecipitation, FLAG-tag, Negative Control, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: Early autophagic and endosomal compartments are involved in the TDP-43-K263E secretion mediated by USP19-WT. a Schematic representation of cellular trafficking and inhibition strategies (pharmacological in blue and siRNAs in red) used in this study to identify potential pathways involved in the TDP-43-K263E secretion mediated by USP19. b-f Evaluation of the TDP-43-K263E secretion mediated by USP19 by FTA in presence of siRNAs control (CT) or siRNAs directed against ATG7 , RAB11A , HRS/HGS , RAB8A or RAB27A . Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from siRNAs CT/targets-treated co-expressing cells using antibodies directed against ATG7, RAB11A, HRS/HGS, RAB8, RAB27A or Flag (for USP19), TDP-43, and GAPDH for loading control. Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of siRNAs CT and targets ATG7 , RAB11A , HRS/HGS , RAB8A and RAB27A are depicted in the right panels of each condition. Data represent mean ± SEM, n = 4 to 6 experiments. Significance was assessed by a Mann–Whitney U test (*p < 0.05)

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Inhibition, Control, Expressing, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: VAMP7 is a modulator of the TDP-43-K263E secretion mediated by USP19-WT. a Evaluation of the TDP-43-K263E secretion mediated by EGFP-VAMP7-WT and Flag-USP19-WT by FTA. Upper panel (secretion): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for USP19), TDP-43, GFP (for VAMP7-WT) and GAPDH for loading control. b Quantification of secreted TDP-43-K263E in presence of GFP-VAMP7-WT or Flag-USP19-WT expression. Data represent mean ± SEM, n = 4 experiments. Significance was assessed by a Mann–Whitney U test, ns = not significant. c Sucrose equilibrium density gradient fractionation of conditioned medium. The 120,000xg pellet (p120K) from conditioned media of TDP-43-K263E + GFP-VAMP7-WT co-expressing cells were fractionated through a 8–60% linear sucrose equilibrium density gradient and fractions (× 15), recovered from the top were analyzed by Western blotting using anti-TDP-43 or anti-CD81 antibody. Fraction density values (g/cm 3 ) are depicted at the bottom panel. d Evaluation of TDP-43-K263E secretion mediated by USP19-WT in presence of EGFP- VAMP7-WT, EGFP-Longin-VAMP7, EGFP-YKT6-WT or EGFP-Longin-YKT6 by FTA. Upper panel (secretion/conditioned medium): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for USP19), TDP-43, GFP (for VAMP7s and YKT6s) and GAPDH for loading control. e Quantification of secreted TDP-43-K263E mediated by Flag- USP19-WT in presence of VAMP7 or YKT6-WT and -Longins expression. Data represent mean ± SEM, n = 4 experiments. Significance was assessed by a Mann–Whitney U test (*p < 0.05), ns = not significant

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY, Fractionation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: DNAJC5/CSPα is a modulator of the misfolded TDP-43-K263E secretion mediated by Flag-USP19-WT. a Evaluation of TDP-43-K263E secretion mediated by 3x-Flag-CSPα-WT or the phospho-deficient CSPα-S10A by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against HA (for HA tagged TDP-43), CSPα and GAPDH for loading control. b Evaluation of the TDP-43-K263E secretion mediated by USP19-WT in presence of CSPα-WT or CSPα-S10A mutant by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for Flag-USP19-WT), TDP-43, CSPα and GAPDH for loading control. c Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of CSPα-WT or the phosphor-deficient CSPα-S10A expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test (**p < 0.001). d Evaluation of TDP-43-K263E secretion mediated by Flag-USP19-WT in presence of siRNAs CT or siRNAs directed against CSPα by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells treated with siRNAs CT or against CSPα using antibodies directed against CSPα, Flag (for Flag-USP19-WT), TDP-43, or GAPDH for loading control. e Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of siRNAs CT or siRNAs CSPα. Data represent mean ± SEM, n = 3 experiments

Article Snippet: The mammalian expression constructs pRK-Flag-USP19 wild-type (USP19 WT , Addgene #78597), USP19 1-1290 (USP19 ΔTM , Addgene #78579), USP19 1-493 (USP19 Nter , Addgene #78581), USP19 494-1318 (USP19 Cter , Addgene #78587) and USP19 C506S (Addgene #78584) were purchased from Addgene and were previously described [ ].

Techniques: Expressing, Western Blot, Control, Mutagenesis, MANN-WHITNEY

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Univariate Cox Model for the Association Between Survival and Clinicopathological Features in GC

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Relative expression of USP19 in GC cell lines and GES1. ( A ) Differential expression of USP19 mRNA in gastric cell lines by real-time PCR assay. ( B ) The differential expression of USP19 protein in gastric cell lines by WB analysis. ( C ) Differential expression and distribution of USP19 protein in gastric cell lines by confocal analysis, Scale bar=20 μm. ( D ) Relative expression of USP19 in paired 212 GC samples and ANTs by IHC staining and analysis by Chi-square test. ( E ) USP19 protein levels in GC tissues and ANTs were analyzed by IHC staining (200× magnification). High or low expression of USP19 protein in ANTs (a and b); high or low expression of USP19 protein in intestinal-type GC (c and d); high or low expression of USP19 protein in diffuse-type GC (e and f), Scale bar=200 μm.

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Expressing, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: The Relationship Between USP19 Expression and Other Clinicopathological Parameters in GC

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Kaplan–Meier survival curves of GC patients according to the clinical features and expression of USP19. ( A ) Patients with high-level USP19 expression showed poor survival. ( B ) TNM stage was a promising indicator of the prognosis of GC patients. ( C ) Patients with intestinal-type GC had better survival than those with diffuse-type GC. ( D ) Low-level USP19 expression predicted better survival in patients with TNM stage III. ( E ) Low-level USP19 expression predicted better survival in patients with diffuse-type GC. ( F ) Low-level USP19 expression predicted better survival in patients with intestinal-type GC.

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Multivariate Cox Model for the Association Between Survival and Clinicopathological Factors in GC

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Functional roles of USP19 in cell models. ( A ) Knockdown of USP19 inhibited SGC7901 cell growth, as determined by MTT assay. Bars: SD; **P<0.01. ( B ) Knockdown of USP19 decreased the level of MMP2 and MMP9 and increased the level of the apoptotic-related protein, cleaved caspase-3, in SGC7901 cells. ( C ) Knockdown of USP19 inhibited colony formation in SGC7901 cells. The data are shown as Mean±SD (n=3). **P<0.01. ( D ) Ectopic expression of USP19 promoted GES1 cell growth, as shown by MTT assay. Bars: SD; **P<0.01. ( E ) Overexpression of USP19 increased the protein level of MMP2 and MMP9 and decreased the level of the apoptotic-related protein, cleaved caspase-3. ( F ) Ectopic expression of USP19 increased colony formation in GES1 cells. The data are shown as Mean±SD (n=3). **P<0.01. All WB experiments were repeated three times.

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Functional Assay, Knockdown, MTT Assay, Expressing, Over Expression

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Effect of USP19 on cell invasion and migration and MMP2/MMP9 enzyme activity. ( A ) Knockdown of USP19 reduced SGC7901 cell invasion. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. ( B ) Knockdown of USP19 reduced the migration of SGC7901 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. ( C ) SGC7901 cells were transfected with ShUSP19 or vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9. ( D ) Ectopic expression of USP19 enhanced invasion of GES1 cells. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. ( E ) Ectopic expression of USP19 increased the migration of GES1 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. ( F ) GES1 cells were transfected with USP19-overexpressing plasmid or control vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9.

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Migration, Activity Assay, Knockdown, Wound Healing Assay, Transfection, Plasmid Preparation, Zymography, Expressing, Control

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Knockdown of USP19 reduced gastric carcinogenesis in animal models. Knockdown of USP19 inhibited tumor growth. Five nude mice were injected subcutaneously with 1 × 10 7 cells/mouse for each of the indicated stable cell lines of SGC7901-ShUSP19 or SGC7901-Vector, respectively. Results were presented as tumor volume at the different time points ( C ) and isolated xenografts at day 24 ( A ) with no change in body weight for each group ( B ). **P<0.01. ( D ) Tumors were isolated, fixed, and subjected to IHC assay (400× magnification).

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Knockdown, Injection, Stable Transfection, Plasmid Preparation, Isolation

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Prognostic roles of USP19 was determined in www.kmplot.com and correlation between mRNA levels of USP19 and MMP2/MMP9 in GC dataset from GEPIA Website ( http://gepia2.cancer-pku.cn ). Overall survival curves were plotted for all GC patients (n=876) with ( A ) different levels of USP19 expression; ( B ) Lauren’s classification; ( C and D ) HER2 status, ( E ) Different treatments, 5-FU based chemical therapy, ( F ) Surgery alone. From these data, low level of USP19 expression was correlated with better OS. The mRNA level of USP19 expression was positive association with MMP2/MMP9 expression. ( G) P<0.001 correlation between MMP9 and USP19; ( H ) P=0.062, correlation between MMP2 and USP19. Especially, the moderate correlation was performed between USP19 and MMP9 expression (Spearman’s R=0.24).

Article Snippet: The specific kit for knockdown of USP19 was ordered from the commercial company (OriGene, Ltd.; USP19 Human shRNA Plasmid Kit [Locus ID 10,869], CAT#: TR308463, ordered from https://www. origene.com/catalog/rnai/shrna-plasmids/tr308463/usp19-human-shrna- plasmid-kit- locus- id-10,869 ).

Techniques: Expressing

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: 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: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ER-anchored USP19 promotes the secretion of misfolded TDP-43 in the HEK293T cellular model. a Schematic representation of Flag-USP19 constructs used in this study. The C506 residue represents an essential amino acid residue necessary for the ubiquitin peptidase catalytic activity. CS 1&2 CHORD-containing proteins and STG1, UBL ubiquitin-like, USP ubiquitin-specific peptidase, TM transmembrane domain, ZnF zinc finger, ΔTM deleted for the transmembrane domain. The pink star represents the amino terminal Flag tag. b Schematic representation of the full-length TDP-43 (upper panel). The K263E variant is highlighted in blue. LCD Low Complexity Domain, NLS nuclear localization sequences, NTD N-terminal domain, RRM1&2 RNA recognition motif 1&2. HEK293T cells were transfected with TDP-43-WT or TDP-43-K263E encoding constructs and were visualized by immunofluorescence using anti-TDP-43 antibody. Blue signal corresponds to DAPI for nuclei and green signal to TDP-43. Scale bar is 10 µm. c Immunoblotting of sarkosyl soluble supernatant (Sark-sol) and sarkosyl insoluble pellet (Sark-ins) fractions isolated from control HEK293T cells (lanes 1 and 4), or cells expressing TDP-43-WT (lanes 2 and 5) or TDP-43-K263E (lanes 3 and 6) using antibodies directed against TDP-43 and GAPDH as loading control. d Evaluation of misfolded TDP-43 secretion by filter trap assay (FTA). Presence of misfolded TDP-43 in conditioned media from HEK293T cells overexpressing TDP-43-WT and TDP-43-K263E and the different Flag-USP19 or the empty vector (negative control) was monitored by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using anti-Flag (for USP19), -TDP-43 and -GAPDH antibodies for loading control. e Quantification of secreted TDP-43 upon USP19 expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test ( **p < 0.001 ). f USP19-WT or USP19-ΔTM and TDP-43-K263E overexpression does not affect plasma membrane permeability and cell viability. HEK293T cells transfected with the indicated plasmids were stained with trypan blue and counted. Data represent mean ± SEM, n = 3 experiments

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Construct, Residue, Ubiquitin Proteomics, Activity Assay, FLAG-tag, Variant Assay, Transfection, Immunofluorescence, Western Blot, Isolation, Control, Expressing, TRAP Assay, Plasmid Preparation, Negative Control, MANN-WHITNEY, Over Expression, Clinical Proteomics, Membrane, Permeability, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ubiquitin peptidase activity is essential for TDP-43-K263E secretion in USP19 context. a Schematic representation of Flag-USP19 constructs. The USP19-C506S corresponds to an ER-anchored but ubiquitin peptidase catalytically inactive mutant. b Ubiquitination level in cell lysates of co-expressing cells. Immunoblotting of cell lysates from TDP-43-K263E and the indicated Flag-USP19 co-expressing cell using antibodies directed against Ubiquitin or GAPDH as loading control. c Evaluation of misfolded TDP-43 secretion by FTA. Presence of misfolded TDP-43 in conditioned media from HEK293T cells overexpressing the TDP-43-K263E, the WT, the ΔTM or the C506S Flag-USP19, was monitored by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using anti-Flag (for USP19), -TDP-43 and -GAPDH antibodies for loading control. d Quantification of secreted TDP-43-K263E upon USP19s expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test (** p < 0.001). e TDP-43-K263E deubiquitination is promoted by USP19-WT. Lysates from HEK293T cells co-expressing Myc-TDP-43-K263E and HA-Ubiquitin-WT with Flag-USP19-C506S (ubiquitin peptidase deficient mutant; lane 1) or Flag-USP19-WT (lane 2) were immunoprecipitated with anti-Myc and immunoblotted with anti-HA and anti-Myc. Whole cell lysates (WCL; 5 μg/input) from co-expressing cells were immunoblotted using anti-Myc, anti-Flag and anti-GAPDH for loading control. Asterisks correspond to heavy and light chains of immunoglobulins used during the immunoprecipitation

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Ubiquitin Proteomics, Activity Assay, Construct, Mutagenesis, Expressing, Western Blot, Control, MANN-WHITNEY, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: The ER-anchored USP19 promotes the secretion of free TDP-43-K263E fibrils in the conditioned medium. a Transmission of misfolded HA-TDP-43-K263E to naïve HEK293T cells. Naive HEK293T cells were cocultured with conditioned media from HEK293T co-expressing cells Flag-USP19-WT or − ΔTM with HA-TDP-43-K263E during 72 h. b After 3 days, cells were washed and analyzed by Western blotting using anti-HA and anti-GAPDH as loading control. c Quantification of n = 5 independent experiments. Significance was assessed by a Mann–Whitney U test (**p < 0.001). d Aggregated TDP-43 levels in conditioned media from HEK293T cells overexpressing TDP-43-K263E and the Flag-USP19 WT and ΔTM were precleared to eliminate cellular debris and ultracentrifuged at 120,000 g pellet (p120K pellet) and analyzed by immunoblotting (lanes 3 and 4) using antibody directed against TDP-43. Cellular lysates (lanes 1 and 2) were analyzed by Western blotting and probed by antibodies directed against TDP-43, Flag and GAPDH (loading control). e Sucrose equilibrium density gradient fractionation of conditioned medium. The p120K pellet was fractionated through a linear sucrose equilibrium density gradient 9–60% and fractions (× 15), recovered from the top of the gradient, were analyzed by Western blotting using anti-TDP-43 or anti-CD81 and anti-CD63 antibodies for extracellular vesicles markers. Fraction density values (g/cm 3 ) are depicted at the bottom panel. Whole cell lysate (WCL) of co-expressing cells was immunoblotted in parallel using the anti-TDP-43, anti-CD63 and anti-CD81. The bottom panel corresponds to the TCE staining of the SDS-PAGE gel. f Immunogold electron microscopy (IEM) of TDP-43-K263E positive fractions. Positive fractions (10–12) containing the TDP-43-K263E were pooled and analyzed by IEM using anti-TDP-43 labelled with a secondary antibody coupled with 10 nm gold particle. Amorphous and fibrillar structures were labelled (red arrows). Scale bars are 20 and 50 nm

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Transmission Assay, Expressing, Western Blot, Control, MANN-WHITNEY, Fractionation, Staining, SDS Page, Electron Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: Transmission electron microscopy (TEM) and RNA sequencing analyses point out ER alterations in TDP-43-K263E and USP19-WT co-expressing cells. a Ultrastructural analyses. TEM of TDP-43-K263E + UPS19-WT (panel i); TDP-43-K263E (panel ii); TDP-43-K263E + USP19-ΔTM (panel iii) and untransfected cells (UT; panel iv as negative control). Black arrows indicate dilated endoplasmic reticulum (ER) accumulation (panels i and v; v-right corresponds to a higher magnification of panel v). White asterisk indicates cytoplasmic aggregates (panel ii). White arrows indicate compact electron dense structures (CEDS; panel iii). Red arrows indicate mitochondria in close contacts with ER (panels v-right to vii). Yellow arrowheads indicate autophagic/endosomal compartments containing ER membrane and dense amorphous structures. (panels v-right, x and xi). Scale bars are 2 and 5 μm. Dotted squares correspond to higher magnification b Volcano plot of differentially expressed genes between TDP-43-K263E/USP19-WT versus TDP-43-K263E/USP19-ΔTM co-expressing cells. Red dots represents upregulated genes (P < 0.05 and Log2FC > 1), blue dots represents downregulated genes (P < 0.05 and Log2FC < -1), and grey dots represent genes that were not differentially expressed. c Bubble plot of the GO Biological process pathway enrichment analysis. The horizontal axis represents the enrichment score (-log10(p-value)), while the vertical axis represents the enriched pathway name. The color scale indicates different thresholds of the p-value, and size of the bubble indicates the number of genes corresponding to each pathway

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Transmission Assay, Electron Microscopy, RNA Sequencing, Expressing, Negative Control, Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: TDP-43-K263E colocalizes with USP19-WT and the KDEL-ER marker and coimmunoprecipitates with USP19-WT. a TDP-43 IEM of HEK293T TDP-43-K263E + Flag-WT-USP19 co-expressing cells. Red arrows indicate the TDP-43 gold particles in close contacts with dilated ER structures in the cytoplasm (panel i) or inside autophagic/endosomal compartments (panels iii and iv). Red arrowheads correspond to free TDP-43 aggregates in intracellular compartments (panel ii). White arrowheads correspond to TDP-43 labelling embedded in amorphous structures present in intracellular autophagic/endosomal compartments (panel iv). b Confocal immunofluorescence imaging of TDP-43-K263E + Flag-USP19-WT co-expressing cells. Co-expressing cells were labelled with antibodies directed against the ER KDEL marker (red), the Flag-USP19-WT (magenta), the HA for TDP-43-K263E (green) and the DAPI (blue) for nuclear staining. Scale bar is 10 μm. c ROI 1–2 are depicted in the merge panel. The plot profiles of the ER-KDEL marker (red) with the Flag-USP19-WT (magenta) and the HA-TDP-43-K263E (green) colocalizations (ROI1 and 2) along the ROI lines were constructed and analyzed using Image J software. White arrowheads show other colocalized signals d TDP-43 coimmunoprecipitates with Flag-USP19-WT. Left panel: co-immunoprecipitation experiment was realized on cell lysates from TDP-43-K263E and Flag-USP19-WT co-expressing cells using mouse antibodies directed against the Flag epitope or an irrelevant SARS-CoV2 IgG antibody as negative control. Right panel: Western blotting of HA-TDP-43-K263E and Flag-USP19-WT co-expressing cell lysates (input) using antibodies directed against Flag (for USP19), HA (for TDP-43) and GAPDH

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Marker, Expressing, Immunofluorescence, Imaging, Staining, Construct, Software, Immunoprecipitation, FLAG-tag, Negative Control, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: Early autophagic and endosomal compartments are involved in the TDP-43-K263E secretion mediated by USP19-WT. a Schematic representation of cellular trafficking and inhibition strategies (pharmacological in blue and siRNAs in red) used in this study to identify potential pathways involved in the TDP-43-K263E secretion mediated by USP19. b-f Evaluation of the TDP-43-K263E secretion mediated by USP19 by FTA in presence of siRNAs control (CT) or siRNAs directed against ATG7 , RAB11A , HRS/HGS , RAB8A or RAB27A . Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from siRNAs CT/targets-treated co-expressing cells using antibodies directed against ATG7, RAB11A, HRS/HGS, RAB8, RAB27A or Flag (for USP19), TDP-43, and GAPDH for loading control. Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of siRNAs CT and targets ATG7 , RAB11A , HRS/HGS , RAB8A and RAB27A are depicted in the right panels of each condition. Data represent mean ± SEM, n = 4 to 6 experiments. Significance was assessed by a Mann–Whitney U test (*p < 0.05)

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Inhibition, Control, Expressing, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: VAMP7 is a modulator of the TDP-43-K263E secretion mediated by USP19-WT. a Evaluation of the TDP-43-K263E secretion mediated by EGFP-VAMP7-WT and Flag-USP19-WT by FTA. Upper panel (secretion): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for USP19), TDP-43, GFP (for VAMP7-WT) and GAPDH for loading control. b Quantification of secreted TDP-43-K263E in presence of GFP-VAMP7-WT or Flag-USP19-WT expression. Data represent mean ± SEM, n = 4 experiments. Significance was assessed by a Mann–Whitney U test, ns = not significant. c Sucrose equilibrium density gradient fractionation of conditioned medium. The 120,000xg pellet (p120K) from conditioned media of TDP-43-K263E + GFP-VAMP7-WT co-expressing cells were fractionated through a 8–60% linear sucrose equilibrium density gradient and fractions (× 15), recovered from the top were analyzed by Western blotting using anti-TDP-43 or anti-CD81 antibody. Fraction density values (g/cm 3 ) are depicted at the bottom panel. d Evaluation of TDP-43-K263E secretion mediated by USP19-WT in presence of EGFP- VAMP7-WT, EGFP-Longin-VAMP7, EGFP-YKT6-WT or EGFP-Longin-YKT6 by FTA. Upper panel (secretion/conditioned medium): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for USP19), TDP-43, GFP (for VAMP7s and YKT6s) and GAPDH for loading control. e Quantification of secreted TDP-43-K263E mediated by Flag- USP19-WT in presence of VAMP7 or YKT6-WT and -Longins expression. Data represent mean ± SEM, n = 4 experiments. Significance was assessed by a Mann–Whitney U test (*p < 0.05), ns = not significant

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY, Fractionation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19

doi: 10.1007/s00018-025-05589-w

Figure Lengend Snippet: DNAJC5/CSPα is a modulator of the misfolded TDP-43-K263E secretion mediated by Flag-USP19-WT. a Evaluation of TDP-43-K263E secretion mediated by 3x-Flag-CSPα-WT or the phospho-deficient CSPα-S10A by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against HA (for HA tagged TDP-43), CSPα and GAPDH for loading control. b Evaluation of the TDP-43-K263E secretion mediated by USP19-WT in presence of CSPα-WT or CSPα-S10A mutant by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells using antibodies directed against Flag (for Flag-USP19-WT), TDP-43, CSPα and GAPDH for loading control. c Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of CSPα-WT or the phosphor-deficient CSPα-S10A expression. Data represent mean ± SEM, n = 5 experiments. Significance was assessed by a Mann–Whitney U test (**p < 0.001). d Evaluation of TDP-43-K263E secretion mediated by Flag-USP19-WT in presence of siRNAs CT or siRNAs directed against CSPα by FTA. Upper panel (secretion/conditioned media): nitrocellulose membranes were probed with an antibody directed against TDP-43. Lower panel (cell expression): Western blotting of cell lysates from co-expressing cells treated with siRNAs CT or against CSPα using antibodies directed against CSPα, Flag (for Flag-USP19-WT), TDP-43, or GAPDH for loading control. e Quantification of secreted TDP-43-K263E mediated by USP19-WT in presence of siRNAs CT or siRNAs CSPα. Data represent mean ± SEM, n = 3 experiments

Article Snippet: The USP19 sgRNA1 and sgRNA2 used for USP19 knockout in HEK293T cells were provided by Yihong Ye (Addgene plasmids #78585 and #78586).

Techniques: Expressing, Western Blot, Control, Mutagenesis, MANN-WHITNEY