lentiviral human atxn3 shrna sequences  (Addgene inc)

Journal: Scientific Reports

Article Title: ATXN3 deubiquitinates ZEB1 and facilitates epithelial–mesenchymal transition in glioblastoma

doi: 10.1038/s41598-025-92317-w

Figure Lengend Snippet: ATXN3 is highly expressed in GBM tissues and cell lines. ( A,B ) The expression levels of ATXN3 mRNA ( A ) and protein ( B ) were analyzed in normal brain tissue (NBT) and gliomas tissues (grades I–IV) (* P < 0.05, *** P < 0.001). ( C ) The expression of ATXN3 was evaluated in NBT and glioma tissues (grades I–IV) using immunohistochemical (IHC) staining. ( D ) Western blotting analysis of ATXN3 protein levels in normal human astrocyte (NHA) and several glioma cell lines. The lower panel is quantification results (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Two non-overlapping lentiviral human ATXN3 shRNA sequences (shATXN3#1 sequence: 5ʹ-CGTCGGTTGTAGGACTAAATA-3ʹ; shATXN3#2 sequences: 5ʹ-GCAGGGCTATTCAGCTAAGTA-3ʹ) were cloned into pLKO.1/U6 (Addgene).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Western Blot

Journal: Scientific Reports

Article Title: ATXN3 deubiquitinates ZEB1 and facilitates epithelial–mesenchymal transition in glioblastoma

doi: 10.1038/s41598-025-92317-w

Figure Lengend Snippet: ATXN3 facilitates the invasion and migration of GBM cells by promoting EMT. ( A ) Western blotting showing ATXN3 protein levels in U251 and LN229 cells transfected with two independent ATXN3-specific shRNAs (shATXN3) or a control shRNA (shCtrl). ( B,C ) Representative images of transwell invasion assay ( B ) and migration assay ( C ) using U251 and LN229 cells transfected with shATXN3#1, shATXN3#2 or shCtrl. Quantification of transwell invasion assay and migration assay are shown (** P < 0.01, *** P < 0.001). ( D ) CCK-8 assays showing the growth rate of 2 GBM cells after indicated treatments (** P < 0.01). ( E ) Western blotting showing EMT-associated protein expression levels in ATXN3-depleted U251 and LN229 cells. ( F ) Western blotting showing ATXN3 protein levels in HS683 and SW1783 cells transfected with ATXN3 or vector control. The lower panel is quantification results (*** P < 0.001). ( G,H ) Representative images of transwell invasion assay ( H ) and migration assay ( G ) using HS683 and SW1783 cells transfected with ATXN3 or vector control. Quantification of transwell invasion assay and migration assay are shown (** P < 0.01, *** P < 0.001). ( I ) CCK-8 assays showing the growth rate of HS683 and SW1783 cells after ATXN3 overexpression (* P < 0.05, ** P < 0.01). ( J ) Western blotting showing EMT-associated protein expression levels in ATXN3-overexpressed HS683 and SW1783 cells. The right panel is quantification results (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Two non-overlapping lentiviral human ATXN3 shRNA sequences (shATXN3#1 sequence: 5ʹ-CGTCGGTTGTAGGACTAAATA-3ʹ; shATXN3#2 sequences: 5ʹ-GCAGGGCTATTCAGCTAAGTA-3ʹ) were cloned into pLKO.1/U6 (Addgene).

Techniques: Migration, Western Blot, Transfection, Control, shRNA, Transwell Invasion Assay, CCK-8 Assay, Expressing, Plasmid Preparation, Over Expression

Journal: Scientific Reports

Article Title: ATXN3 deubiquitinates ZEB1 and facilitates epithelial–mesenchymal transition in glioblastoma

doi: 10.1038/s41598-025-92317-w

Figure Lengend Snippet: ATXN3 maintains the stability of ZEB1. ( A ) Plasmids shATXN3#1, shATXN3#2 or shCtrl were stably transduced into U251 and LN229 cells. The expression levels of ATXN3, ZEB1, ZEB2, Snail1, SLUG and Twist1 were examined by western blotting using the indicated antibodies. ( B ) QRT-PCR analysis of ATXN3 and ZEB1 mRNA levels in U251 and LN229 cells transduced with shATXN3#1, shATXN3#2, or shCtrl. ( C ) Western blotting showing ZEB1 protein levels in U251 and LN229 cells transfected with Flag-tagged ATXN3 wild-type (WT) and catalytically inactive C14A mutant plasmids. The right panel is quantification results (n.s. = not significant, ** P < 0.01). ( D ) U251 and LN229 cells transduced with shATXN3#1, shATXN3#2, or shCtrl were treated with the proteasome inhibitor MG132 (20 μM, 8 h) or left untreated, followed by analysis of ATXN3 and ZEB1 protein levels. ( E–H ) U251 ( E,F ) and LN229 ( G,H ) cells transduced with shATXN3#1 or shCtrl were treated with 50 μg/ml cycloheximide for various time intervals. ZEB1 protein levels were assessed by Western blotting, and ZEB1 expression was quantified relative to GAPDH (** P < 0.01).

Article Snippet: Two non-overlapping lentiviral human ATXN3 shRNA sequences (shATXN3#1 sequence: 5ʹ-CGTCGGTTGTAGGACTAAATA-3ʹ; shATXN3#2 sequences: 5ʹ-GCAGGGCTATTCAGCTAAGTA-3ʹ) were cloned into pLKO.1/U6 (Addgene).

Techniques: Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR, Transduction, Transfection, Mutagenesis

Journal: Scientific Reports

Article Title: ATXN3 deubiquitinates ZEB1 and facilitates epithelial–mesenchymal transition in glioblastoma

doi: 10.1038/s41598-025-92317-w

Figure Lengend Snippet: ATXN3 interacts with ZEB1 and removes its ubiquitin moieties. ( A ) HEK293T cells were transduced with either Myc-ZEB1 alone or in combination with Flag-tagged ATXN3 WT or the mutant C14A. Subsequently, cell lysates were immunoprecipitated using an anti-Flag antibody, and the immunoprecipitates were analyzed by Western blotting with the specified antibodies. ( B ) Reciprocal co-immunoprecipitation of endogenous ATXN3 and ZEB1 in U251 and LN229 cells. ( C ) Representative confocal images illustrate the colocalization of ATXN3 (green) and ZEB1 (red) in U251 and LN229 cells. Scale bars = 10 µm. ( D ) Plasmids Myc-ZEB1, HA-ubiquitin (HA-Ub), and either Flag-tagged ATXN3 WT or the C14A mutant were co-transduced into MG132-treated HEK293T cells. Following this, the cells were lysed for immunoprecipitation with an anti-Myc antibody and analyzed via Western blotting. ( E ) U251 and LN229 cells transfected with HA-Ub and either shATXN3#1 or shCtrl were lysed for immunoprecipitation with an anti-ZEB1 antibody and subsequently analyzed by Western blotting. ( F ) Myc-tagged ubiquitinated ZEB1 and Flag-tagged ATXN3 WT or C14A mutant were purified from HEK293T cells. Ubiquitinated ZEB1 was then incubated with either ATXN3 WT or C14A mutant, followed by Western blotting with the indicated antibodies.

Article Snippet: Two non-overlapping lentiviral human ATXN3 shRNA sequences (shATXN3#1 sequence: 5ʹ-CGTCGGTTGTAGGACTAAATA-3ʹ; shATXN3#2 sequences: 5ʹ-GCAGGGCTATTCAGCTAAGTA-3ʹ) were cloned into pLKO.1/U6 (Addgene).

Techniques: Ubiquitin Proteomics, Transduction, Mutagenesis, Immunoprecipitation, Western Blot, Transfection, Purification, Incubation

Journal: Scientific Reports

Article Title: ATXN3 deubiquitinates ZEB1 and facilitates epithelial–mesenchymal transition in glioblastoma

doi: 10.1038/s41598-025-92317-w

Figure Lengend Snippet: ZEB1 is essential for ATXN3-driven invasion, migration, proliferation, and tumorigenic capacity in GBM cells. ( A ) Western blotting demonstrating the protein expression levels of ATXN3, ZEB1, and EMT-related proteins in U251 and LN229 cells co-transfected with shATXN3#1/shCtrl or ZEB1/vector control. B-C, Representative images of transwell invasion assay ( B ) and migration assay ( C ) in U251 and LN229 cells transfected with shATXN3#1 and either ZEB1 or empty vector. Quantification of transwell invasion assay ( B ) and migration assay ( C ) are shown (** P < 0.01, *** P < 0.001). ( D ) CCK-8 assays showing the growth rate of U251 and LN229 cells after indicated treatment (** P < 0.01, *** P < 0.001). ( E–G ) Tumor xenografts were created by subcutaneously inoculating U251 cells transfected with indicated plasmids into the right armpit of nude mice. Tumor sizes were measured every 10 days using a vernier caliper, after 30 days, the mice were sacrificed, and the tumors were excised and weighed (n = 6) (** P < 0.01, *** P < 0.001).

Article Snippet: Two non-overlapping lentiviral human ATXN3 shRNA sequences (shATXN3#1 sequence: 5ʹ-CGTCGGTTGTAGGACTAAATA-3ʹ; shATXN3#2 sequences: 5ʹ-GCAGGGCTATTCAGCTAAGTA-3ʹ) were cloned into pLKO.1/U6 (Addgene).

Techniques: Migration, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Transwell Invasion Assay, CCK-8 Assay

Journal: Scientific Reports

Article Title: ATXN3 deubiquitinates ZEB1 and facilitates epithelial–mesenchymal transition in glioblastoma

doi: 10.1038/s41598-025-92317-w

Figure Lengend Snippet: ATXN3 is positively correlated with ZEB1 protein levels and serves as a predictor of poor prognosis in GBM. ( A ) Representative immunohistochemical images showing the expression levels of ATXN3 and ZEB1 in two GBM specimens. ( B ) The correlation between ATXN3 and ZEB1 expression levels was assessed using the Spearman correlation test in 64 GBM specimens. ( C,D ) Kaplan–Meier analysis of progression-free ( C ) and overall survival ( D ) in GBM patients with high (n = 32) or low (n = 32) ATXN3 expression. ( E ) Schematic illustration of the main findings of this study, which indicates that ATXN3 stabilizes ZEB1 expression and promotes EMT in GBM.

Article Snippet: Two non-overlapping lentiviral human ATXN3 shRNA sequences (shATXN3#1 sequence: 5ʹ-CGTCGGTTGTAGGACTAAATA-3ʹ; shATXN3#2 sequences: 5ʹ-GCAGGGCTATTCAGCTAAGTA-3ʹ) were cloned into pLKO.1/U6 (Addgene).

Techniques: Immunohistochemical staining, Expressing

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of VCP cofactors and their proposed functions

Article Snippet: ATXN3 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR302905.

Techniques: Membrane

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: VCP cofactors differentially regulate tau seeding. VCP cofactors were either knocked out via CRISPR/Cas9 ( A-D ) or knocked down via siRNA ( E–G ) in v2L biosensors prior to exposure to increasing amounts of tau fibrils. A Knockout of FAF2 increased tau seeding whereas knockout of B ATXN3, C NSFL1C, and D UBE4B reduced tau seeding. P values: FAF2 (*** 0.0001, **** < 0.0001); ATXN3 (**** < 0.0001); NSFL1C (*** 0.0002); UBE4B (**** < 0.0001). E Knockdown of NGLY1, F NPLOC4, and G OTUB1, decreased tau seeding. P values: NGLY1(**** < 0.0001); NPLOC4 (**** < 0.0001); OTUB1 (*** 0.0004, **** < 0.0001, *** 0.0001). Graphs are representative of n = 3 independent experiments, with each data point derived from technical triplicate. Error bars represent S.D. Some error bars are too small to be visible. H Cofactor KO did not affect tau uptake. P values: ns = 0.9819, 0.9988, 0.9956, 0.9928, in order of bars on the graph. I Cofactor KD did not affect tau uptake. P values: ns = 0.9795, 0.1856, 0.3928, in order of bars on the graph. Error bars represent S.E.M ( n = 3). One-Way ANOVA with a 95% confidence interval

Article Snippet: ATXN3 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR302905.

Techniques: CRISPR, Knock-Out, Knockdown, Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of Reagents

Article Snippet: ATXN3 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR302905.

Techniques: Protease Inhibitor, Cell Culture, Transfection, Magnetic Beads, Sequencing, Modification

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of Reagents

Article Snippet: ATXN3 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR302905.

Techniques: Protease Inhibitor, Cell Culture, Transfection, Magnetic Beads, Sequencing, Modification

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of Reagents

Article Snippet: ATXN3 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR302905.

Techniques: Protease Inhibitor, Cell Culture, Transfection, Magnetic Beads, Sequencing, Modification