Journal: The Journal of Neuroscience

Article Title: Calpain Inhibition Is Protective in Machado–Joseph Disease Zebrafish Due to Induction of Autophagy

doi: 10.1523/JNEUROSCI.1142-17.2017

Figure Lengend Snippet: Real-time PCR revealed that changes in human ataxin-3 and p62 levels following calpeptin treatment were not present at an mRNA level. A, Real-time PCR of human ATXN3 mRNA levels in control (nontransgenic, n = 3), EGFP-ataxin-3-23Q (n = 3) and EGFP-ataxin-3-84Q (with and without 50 μm calpeptin treatment, n = 3 and 4, respectively) revealed similar human ataxin-3 levels in all transgenic MJD zebrafish samples. All transgenic ataxin-3 zebrafish groups had significantly higher human ATXN3 levels than control (∧p < 0.041), with no significant decrease in human ATXN3 mRNA levels in EGFP-ataxin-3-84Q larva receiving calpeptin (50 μm) treatment. B, Real-time PCR of p62 mRNA levels in control, EGFP-ataxin-3-23Q and EGFP-ataxin-3-84Q (with and without calpeptin treatment) samples revealed that EGFP-ataxin-3-84Q zebrafish have higher p62 mRNA levels than control and EGFP-ataxin-3-23Q larva; #p < 0.031. Calpeptin treatment did not effect the p62 mRNA levels of EGFP-ataxin-3-84Q zebrafish. n values were the same as in A. Error bars represent mean ± SEM.

Article Snippet: Predesigned TaqMan Gene Expression Assays were used containing primers specific for transgenic ATXN3 (Assay ID Hs00245261_m1), and primers for atp5b (Assay ID Dr0342455_m1), and eef1a (Assay IDDr03432748_m1) as reference genes.

Techniques: Real-time Polymerase Chain Reaction, Control, Transgenic Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: The spot annotated within the dashed box (absent in the control IP) was identified as human ATXN3 by subsequent MS/MS analysis .

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Control, Tandem Mass Spectroscopy

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: HEK-293 cells were transfected with either PNKP siRNA or ATXN3 siRNA or control siRNA and the nuclear extracts (1 mg) prepared from those cells were IP’d with anti-PNKP Ab (BioBharati Life Science Pvt. Ltd, Kolkata, India; 2A ) or anti-ATXN3 Ab (Proteintech, 2B ) or with IgG as a control, and tested for the presence of PNKP- and ATXN3-associated proteins with Abs to the proteins shown on the right. (C) Detection of PNKP’s interaction with ATXN3 in SH-SY5Y cells by proximity ligation assays using a Duolink kit (Olink Bioscience, Uppsala, Sweden). Nuclei were counterstained with DAPI (blue). ATXN3-depleted (by siRNA) cells were used as a control to show the specificity of the interaction (middle panel). Right panel, Non-specific Ab (IgG) control (D) GST-PNKP pull-down of ATXN3 (WT and mutant) using purified GST-tagged full-length or three domains (FHA-, Kinase- and Phosphatase-domain) of PNKP, probed with anti-ATXN3. Bottom panel, Coomassie-stained gel of the corresponding PNKP domains, a second gel run in parallel.

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Transfection, Control, Ligation, Mutagenesis, Purification, Staining

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) A 32 P-labeled 3’-phosphate-containing oligo substrate (5 pmol) was incubated at 37°C for 10 min in buffer A (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol and 0.1 μg/μl acetylated BSA) with PNKP alone (50 fmol, ln 2) or ATXN3-Q29 alone (400 fmol, ln 8) or PNKP plus increasing amounts of Q29 (25, 50, 100, 200 and 400 fmol; lns 3–7). Ln 1, substrate only. Generation of the 32 P-labelled 3’P-containing oligo substrate was described previously . (B) Effect of mutant ATXN3 (Q72) on PNKP’s 3’-phosphatase activity. 32 P-labelled 3’-phosphate-containing oligo substrate (2.5 pmol) was incubated with PNKP at 37°C for 20 min alone (50 fmol, ln 2), ATXN3-Q72 alone (200 fmol, ln 3), PNKP (50 fmol) plus increasing amounts of Q72 (50, 100 and 200 fmol, lns 4–6). Ln 1, no protein, substrate only. Quantitation of the products (released phosphate) is represented in the histogram (lower panel), with the activity of PNKP alone arbitrarily set as 1 (n = 3, ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Labeling, Incubation, Mutagenesis, Activity Assay, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) Western blot analysis showing the level of expression of ATXN3 in SH-SY5Y cells stably expressing control vs. ATXN3-shRNA. Lamin B was used as a loading control. (B) A representative gel (n = 3) showing the 3’-phosphatase activity in the nuclear extract (200 ng) of control (ln 3) and ATXN3-depleted cells (ln 4). Lns 5–8, increasing amounts (10, 25, 50 and 100 fmol) of purified ATXN3 (WT) were added back to ATXN3-depleted nuclear extract. Quantitation of the products is represented in the histogram (n = 3, ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Western Blot, Expressing, Stable Transfection, Control, shRNA, Activity Assay, Purification, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: Long amplicon qPCR (LA-QPCR) was used to evaluate genomic DNA SB levels in control vs. ATXN3-depleted HEK-293 cells. Representative gel showing PCR-amplified fragments of the HPRT (left panel) and POLB (right panel) genes. Amplification of each large fragment (upper panels) was normalized to that of a small fragment of the corresponding gene (bottom panels) and the data were expressed as lesion frequency/10 Kb DNA as described previously . Histograms represent the DNA damage quantitation for control vs ATXN3-depleted cells (n = 3, ** = P< 0.01). Error bars indicate standard error of means.

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Amplification, Control, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) Upper panel, 32 P-labelled 3’-phosphate-containing oligo substrate (2.5 pmol) was incubated with the NE (250 ng) isolated from rat mesenchymal (CSM14.1) cells conditionally expressing vector alone (lane 1), WT (Q-23, lane 2) or mutant ATXN3 (Q-70, lane 3). Ln 4, no protein. S: substrate, P: product (released 32 P i ). Middle panel: Western analysis of the corresponding NE (10μg) showing PNKP expression. GAPDH (Ab from Genetex Inc.) was used as loading control. Bottom panel: quantitation of the products (P) is represented in a histogram. (n = 3, ** = P< 0.01) (B) Comet assays showing the accumulation of DNA SBs in mutant (Q-70) ATXN3 cells. (C) Comparative (WT vs. SCA3 mice) 3’-phosphatase activity in the NEs from four different brain regions. 7.5 pmol of 3’-P-containing substrate was incubated at 37°C for 10 min with WT or transgenic mouse brain NE (200 ng; PN-Pontine Nuclei, SN-Substantia Nigra, DCN-Deep Cerebellar Nuclei, and HP-Hippocampus). Quantitation of the products is shown in the histogram with the age-matched WT arbitrarily set as 1 (n = 5; ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Incubation, Isolation, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Control, Quantitation Assay, Activity Assay, Transgenic Assay

Journal: Acta neuropathologica

Article Title: Insulin‑like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

doi: 10.1007/s00401-020-02183-1

Figure Lengend Snippet: overexpression reduced the levels of huntingtin in MSNs derived from HD patients. a HEK293T cells were co-transfected with full length (FL) HttQ103-myc and IGF2 or empty vector (Mock). 24 h later, FL HttQ103-myc levels were analyzed in whole cell extracts by western blot analysis using an anti-Htt antibody. The arrow indicates the migration of full-length huntingtin. b HEK293T cells were co-transfected with FL HttQ103-myc and IGF2 or empty vector (Mock). After 24 h, FL HttQ103-myc was detected in cell lysates by dot blot. Statistically significant differences detected by two-tailed unpaired t test (***p < 0.001). c Schematic representation of the differentiation process to generate mature medium spiny neurons (MSNs) from induced human pluripotent stem cells (iPSCs), showing cell morphology using contrast phase microscopy. d Confirmation of the expression of GABAergic (DARPP-32) and neuronal (MAP2) markers in control and HD patients MSNs using immunofluorescence. e MSNs from control subjects and HD patients were transduced with adeno-associated viral (AAV) vectors expressing IGF2 or empty vector (Mock). 17 days later, huntingtin protein levels were measured in total protein extracts by western blot using anti-Htt antibody. Vinculin expression was monitored as loading control (lower panel). Values represent the mean and SEM of three independent experiments. Statistically significant differences detected by one-way ANOVA (*p < 0.05). f Schematic representation of the generation of neuronal cultures generated from SCA-derived iPSCs. g Ataxin 3 levels were monitored in control and SCA3 patient fibroblasts using western blot analysis. “Asterisk” represents the mutant protein. h Differentiated SCA3 iPSC-derived neurons at 90 days post-differentiation were stained for βIII-tubulin neuronal marker. Scale bars: 50 μm. i Neurons were treated with 0.1 mM L-glutamate for 1 h. Then, detergent insoluble protein extracts were generated in triton X100 (TX-100) and pellets were resuspended in SDS to yield an SDS-soluble fraction (SDS) and SDS-insoluble fraction that was then dissolved in formic acid (FA). Ataxin 3 levels were monitored in all fractions using western blots analysis (*-expanded allele). Two independent experiments were performed. j SCA3 cells were stimulated with IGF2-conditioned media for indicated time points followed by stimulation with 0.1 mM L-glutamate followed by western blot analysis as described in (i). GAPDH levels were determined as loading control and βIII-tubulin as a neuronal marker

Article Snippet: Samples were probed with primary antibodies: Ataxin-3 (Aviva Systems Biology; Cat#ARP50507_P050), βIII-tubulin (Abcam; Cat#ab7751) and GAPDH (Fitzgerald; Cat#10R-G109A).

Techniques: Over Expression, Derivative Assay, Transfection, Plasmid Preparation, Western Blot, Migration, Dot Blot, Two Tailed Test, Microscopy, Expressing, Immunofluorescence, Transduction, Generated, Mutagenesis, Staining, Marker

Journal: Life Medicine

Article Title: CRISPR/Cas9-mediated genetic correction reverses spinocerebellar ataxia 3 disease-associated phenotypes in differentiated cerebellar neurons

doi: 10.1093/lifemedi/lnac020

Figure Lengend Snippet: CRISPR/Cas9-mediated genetic correction of SCA3-specific iPSCs using an “intron-based strategy”. (A) A schematic view of a genetic correction strategy of the mutant ATXN3 allele in SCA3 iPSCs. Gray boxes represent exons at the ATXN3 locus. The mutant ATXN3 allele was corrected using CRISPR/Cas9-mediated HDR by adjusting the pathological 80 or 78 CAG repeats (red box) to a normal 13 CAG repeats (green box). The Cas9 protein-blocking mutations (CBMs) (GG to CC) at the PAM region (magenta) were introduced in the donor arms, preventing undesired editing of the target site (blue). P1 and P2 represent PCR primers for amplification. (B) Procedure of targeted correction of SCA3 iPSCs. FACS, fluorescence-activated cell sorting. (C) The ratio of GFP-positive iPS single cells by FACS after 1 day of transfection with the pCas9-GFP plasmid, sgRNA expression vectors and donor. (D and E) PCR-based screen of genetically corrected iPSC clones after gene targeting in Pa1 and Pa2-SCA3 iPSCs. The corrected clones and represented are indicated in green. An uncorrected clone with a large-scale deletion is indicated in red. M, marker. (F and G) Sequencing results of the genetically corrected clones (C111, C118, C206, C-21) near the region of CAG repeats, including the sgRNA target sites (blue) and PAM (magenta). (CAG)n, CAG repeat number; red triangles, cleavage sites; Δ10, delete 10 base pairs; +A, insert an adenine. (H) Western blot analysis indicated that C111, C118, C206, C-21 and WT iPSCs only have a normally sized ataxin-3 protein detected by the ATXN3-specific and polyQ-specific antibody 1C2, when compared to Pa1 and Pa2-SCA3 iPSCs.

Article Snippet: The other rabbit anti-ATXN3 polyclonal antibody (TA327022) was purchased from Origene.

Techniques: CRISPR, Mutagenesis, Blocking Assay, Amplification, Fluorescence, FACS, Transfection, Plasmid Preparation, Expressing, Clone Assay, Marker, Sequencing, Western Blot

Journal: Life Medicine

Article Title: CRISPR/Cas9-mediated genetic correction reverses spinocerebellar ataxia 3 disease-associated phenotypes in differentiated cerebellar neurons

doi: 10.1093/lifemedi/lnac020

Figure Lengend Snippet: CRISPR/Cas9-mediated genetic correction of SCA3-specific iPSCs using an “exon-based strategy”. (A) The schematic view of the “exon-based strategy”. Donor-608 was used as a template with homologous arms of 81 bp on the left and 406 bp on the right. CBMs (TTCT to CTCC and GAC to GAT) (fuchsia) were introduced to prevent undesired editing of the target site (blue). P3, P4, PCR Primer pair. (B) The overview of CBMs designed in the exon-based strategy, which introduced TTCT to CTCC in the intron and GAC to GAT in the exon, leaving a same-sense mutation but an unrecognized PAM region by Cas9 in the exon. Fuchsia shades represent the sites of the CBMs. Target sites and PAM region are indicated in blue and magenta, respectively. Red triangles, cleavage sites. (C and D) PCR screening of iPS clones with P3 and P4 primer pairs. Corrected clones represented are indicated in green. M, marker. (E) Examination of donor DNA random integration into the chromosomes by PCR using primer pair D608-T. (F) Sequencing analysis of the genetically corrected clone 1A. The CBMs integrated into the genome by HDR are in red. (G) Sequencing results revealed that clone 1A was corrected by adjusting both the mutant CAG allele (CAG80) and WT allele (CAG23) to 13-CAG repeats. Red triangles, cleavage sites; fuchsia shades, the sites of the CBMs. (H) Western blot analysis indicated that clone 1A and WT iPSCs only have a normally sized ataxin-3 protein detected by the ATXN3-specific and polyQ-specific antibody 1C2, when compared to Pa1-SCA3 iPSCs.

Article Snippet: The other rabbit anti-ATXN3 polyclonal antibody (TA327022) was purchased from Origene.

Techniques: CRISPR, Mutagenesis, Clone Assay, Marker, Sequencing, Western Blot

Journal: Life Medicine

Article Title: CRISPR/Cas9-mediated genetic correction reverses spinocerebellar ataxia 3 disease-associated phenotypes in differentiated cerebellar neurons

doi: 10.1093/lifemedi/lnac020

Figure Lengend Snippet: Reversion of ataxin-3 spontaneous aggregation in genetically corrected SCA3 cerebellar neurons. (A) A schematic view of the separation of the cell lysate for investigating the ataxin-3 aggregates. (B-D) Western blot analysis with the ATXN3-specific antibody showed that SDS-insoluble ataxin-3 aggregates appeared in 6-week-differentiated rather than 3-week-differentiated SCA3 cerebellar neurons (C and D), nor in WT (B) and genetically corrected cerebellar neurons (C and D). (E) Ataxin-3 spontaneous aggregation in specific SCA3 cerebellar neurons but not in other cell types such as iPSCs, cerebellar NSCs, pNSCs and pan-neurons derived from pNSCs of SCA3. Strikingly, the hallmark aggregates were not detected in the genetically corrected cells. (F–I) Ataxin-3 aggregation in the presence of the excitatory neurotransmitter l -glutamate (100 μM) or the calpain inhibitor calpeptin (100 μM) in SCA3 cerebellar neurons and pan-neuronal cells (F, H). Remarkably, SDS-insoluble ataxin-3 aggregates were not detected in either the genetically corrected cerebellar neurons or the pan-neuronal cells (G, I). TX100, Triton-X-100; FA, formic acid. n ≥ 3 individual experiments.

Article Snippet: The other rabbit anti-ATXN3 polyclonal antibody (TA327022) was purchased from Origene.

Techniques: Western Blot, Derivative Assay

Journal: Life Medicine

Article Title: CRISPR/Cas9-mediated genetic correction reverses spinocerebellar ataxia 3 disease-associated phenotypes in differentiated cerebellar neurons

doi: 10.1093/lifemedi/lnac020

Figure Lengend Snippet:

Article Snippet: The other rabbit anti-ATXN3 polyclonal antibody (TA327022) was purchased from Origene.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 1. ATXN3 positively regulates IFN-I antiviral efficacy. (A) 293T, HeLa, and A549 cells were transfected with or without FH-ATXN3. Then 293T cells were infected with VSV. HeLa cells were infected with SeV, and A549 cells were infected with H1N1. After 20 h culture, the viral RNA levels were analyzed by qPCR. (B and C) 293T cells transfected with either FH-ATXN3 (B) or shATXN3 (C) were infected with VSV for 20 h, and VSV titers in culture supernatants were determined by a TCID50 assay. (D) 293T cells transfected with FH-ATXN3 were pretreated with or without IFN-a (60 IU/ml) for 20 h. Then cells were infected with VSV-GFP (multiplicity of infection of 0.5) for 20 h. The VSV-GFP signal was detected by fluorescence microscopy. Original magnification 320. (E) VSV-GFP levels in (D) were analyzed by FACS. (F) 293T cells transfected with control shRNAs (shCON) or two shATXN3 were pretreated with IFN-a (60 IU/ml) for 20 h, and then cells were infected with VSV for 24 h. Whole-cell extracts were analyzed by Western blots using the indicated Abs. The protein band density was quantified with ImageJ. (G) HeLa cells transfected with empty vectors, HA- ATXN3-20Q, or HA-ATXN3-79Q were treated as in (D). The viral RNA levels of VSV were analyzed by qPCR. Data are from three experiments [(A–C and G) mean and SD of three independent replicates] or representative of three independent experiments (D–F). *p , 0.05, **p , 0.01, ***p , 0.001. n.s, not significant.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Transfection, Infection, TCID50 Assay, Microscopy, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 2. ATXN3 does not affect the production of IFN-I. (A) 293T cells were transfected with FH-ATXN3, together with IFN-b–luciferase (IFN-b– Luc) and Renilla. The luciferase activity was measured after cells were infected with SeV for 20 h. (B) qPCR analysis of IFN-a mRNA (left) and IFN-b mRNA (right) in HT1080 cells transfected with FH-ATXN3 followed by SeV infection for 6 h. (C) HT1080 cells transfected with shATXN3 were infected with SeV for 12 h, and the mRNA levels of IFN-a and IFN-b were analyzed by qPCR. (D and E) 293T cells were transfected with either FH-ATXN3 (D) or shATXN3 (E) and then infected with VSV for 20 h. IFN-a protein in culture supernatants was analyzed by ELISA. (F) HT1080 cells transfected with or without FH-ATXN3 were infected with VSV for 10 h, and their culture supernatants were collected and used to stimulate another plate of HT1080 cells for 4 h. The mRNA levels of IFIT1 were analyzed by qPCR. Data are from three experiments (mean and SD of three independent replicates). ***p , 0.001. n.s, not significant.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Transfection, Luciferase, Activity Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 3. ATXN3 enhanced the IFN-I–mediated signaling pathway. (A) 293T cells were transfected with empty vectors or FH-ATXN3, together with ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (B) 293T cells were transfected with either control shRNAs (shCON), shATXN3 no. 1, or shATXN3 no. 2, together with ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (C) PC9 cells transfected with either shCON or shATXN3 were treated with IFN-a (1000 IU/ml) for 6 h, and the mRNA levels of IFIT1, ISG15, and ISG54 were analyzed by qPCR. (D) Mouse primary lung cells were infected with shATXN3-packaged lentiviruses and then were stimulated with mIFN-b (300 IU/ml) for 4 h. The mRNA levels of IFIT1 were analyzed by qPCR. (E) 293T cells were transfected with different amounts of FH-ATXN3. Whole-cell lysates were analyzed by indicated Abs. (F) 293T cells transfected with FH-ATXN3 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Whole-cell extracts were subjected to SDS-PAGE and probed with a pY701-STAT1 (p-STAT1) Ab. (G) HepG2 cells transfected with either shCON or two shATXN3 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Immunoblotting was performed as in (F). (H) Mouse primary lung cells were infected as in (D) and then were treated with mIFN-b (300 IU/ml) for 30 min. Immunoblotting was performed as in (F). (I) HeLa cells transfected with shCON and shATXN3 were infected with VSV for 12 h. Whole-cell extracts were analyzed by immunoblotting using the pY701-STAT1 Ab. Data are from three experiments [(A–D) mean and SD of three independent replicates] or representative of three independent exper- iments (E–I). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Transfection, Luciferase, Activity Assay, Control, Infection, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 4. ATXN3 upregulates HDAC3 expression at the protein level. (A, C, and E) 293T cells were transfected with Flag-tagged HDAC1 (A), HDAC2 (C), or HDAC3 (E), together with increased amounts of FH-ATXN3. Whole-cell extracts were subjected to immunoblotting using the indicated Abs. (B, D, and F) 293T cells were transfected with Flag-tagged HDAC1 (B), HDAC2 (D), or HDAC3 (F), together with control shRNAs (shCON) or shATXN3. Protein levels of Flag-HDACs were detected by an anti-Flag Ab. (G) 293T cells were transfected with increased amounts of FH-ATXN3, and the level of en- dogenous HDAC3 was analyzed as indicated. (H) 293T cells were transfected with shCON or shATXN3. Cell lysates were analyzed by Western blots using indicated Abs. Data are representative of three independent experiments.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 5. ATXN3 interacts with HDAC3. (A and B) 293T cells were transfected with Myc-ATXN3 and/or Flag-HDAC3 as indicated. Immunoprecipitation was performed using an anti-Myc Ab (A) or M2 beads (B), followed by immunoblotting as indicated. (C) Endogenous ATXN3 interacts with endogenous HDAC3. HT1080 cells were harvested and endogenous ATXN3 was immunoprecipitated, and then immunoblotting was performed using an anti- HDAC3 Ab. (D) Mouse primary lung cells were infected with VSV for 0, 6, or 12 h. Whole-cell lysates were subjected to immu- noprecipitation with an anti-ATXN3 Ab and then were analyzed by Western blots using the indicated Abs. Data are representative of three independent experiments.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 6. ATXN3 promotes the stability of HDAC3. (A) HeLa cells transfected with control shRNAs (shCON) or two shATXN3 were treated with MG132 (10 mM) for 12 h. Cell lysates were analyzed by Western blots using indicated Abs. (B) 293T cells were transfected with Flag-HDAC3, together with or without FH-ATXN3. Then the cells were treated with CHX (50 mg/ml) for different times. The whole-cell extracts were analyzed by immuno- blotting using the indicated Abs. (C) 293T cells were transfected with Flag-HDAC3, together with either shCON, shATXN3 no. 1, or shATXN3 no. 2. Then the cells were treated with CHX (50 mg/ml) for different times. The whole-cell extracts were analyzed as described in (B). (D) 293T cells transfected with Flag-HDAC3 were pretreated with DMSO or MG132 (10 mM) for 12 h, followed by treatment with CHX (50 mg/ml) for different times as indicated. Whole-cell lysates were analyzed by indicated Abs. Data are representative of three independent experiments.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Transfection, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 7. ATXN3 removes both K48-linked and K63-linked polyubiquitination of HDAC3. (A) 293T cells were transfected with HA-ub, together with Myc-ATXN3 and/or Flag-HDAC3 as indicated. Flag-HDAC3 was immunoprecipitated with M2 beads for 3 h and then immunoblotting was performed as indicated. (B) 293T cells were transfected with Flag-HDAC3 and control shRNAs or shATXN3 as indicated. Then cells were treated with MG132 (10 mM) for 12 h. Cell lysates were subjected to immunoprecipitation with M2 beads and then analyzed by Western blots with an anti-ubiquitin Ab. (C) 293T cells were transfected with HA-ub and two shATXN3 as indicated. Endogenous HDAC3 was immunoprecipitated by an anti-HDAC3 Ab, and the ubiquitination level of HDAC3 was analyzed as indicated. (D) 293T cells were transfected with Flag-HDAC3, together with HA-ub-K48 (R48K) or HA-ub-K63 (R63K) and/or Myc-ATXN3 as indicated. Flag-HDAC3 was immunoprecipitated by M2 beads, and then immunoblotting was performed using indicated Abs. (E) 293T cells were transfected with Flag-HDAC3, together with HA-ub-K48 (R48K) or HA-ub-K63 (R63K) and/or shATXN3. Cells were treated with MG132 for 12 h. Immunoprecipitation was performed using M2 beads, followed by immunoblotting using an anti-HA Ab. Data are representative of three in- dependent experiments.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Ubiquitin Proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 8. ATXN3 regulates IFN-I antiviral response dependent on its deubiquitinase activity. (A) Map of the ATXN3 construction is shown (upper panel). HepG2 cells were transfected with FH-ATXN3 (WT) or ATXN3-C14A/H119D/N134S mutant (TM), and the level of endogenous HDAC3 was analyzed as indicated. (B) 293T cells were transfected with HA-ub, together with Myc-ATXN3-WT or Myc-ATXN3-TM. Endogenous HDAC3 was immunoprecipitated with an anti-HDAC3 Ab, followed by immunoblotting using an anti-HA Ab. (C) HeLa cells transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM were stimulated with IFN-a (1000 IU/ml) for 30 min. Whole-cell lysates were analyzed by indicated Abs. (D) 293T cells were transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM, together with ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (E and F) HeLa cells (E) or 293T cells (F) transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM were infected with H1N1 (E) or VSV (F), the HA protein of H1N1 was detected by Western blots (E), and the VSV mRNA level was analyzed by qPCR. (G) 293T cells transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM were treated with IFN-a (60 IU/ml) for 20 h and then infected with VSV-GFP for 24 h. The VSV-GFP signal was detected by fluorescence microscopy. Original magnification 320. Data are from three experiments [(D and F) mean and SD of three independent replicates] or representative of three independent experiments (A–C, E, and G). *p , 0.05. n.s, not significant.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Activity Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Luciferase, Infection, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3.

doi: 10.4049/jimmunol.1800285

Figure Lengend Snippet: FIGURE 9. ATXN3 promotes IFN-I–mediated signaling and antiviral activity dependent on HDAC3. (A) 293T cells were transfected with either control shRNAs (shCON) or two shHDAC3, together with empty vectors or FH-ATXN3. Then cells were stimulated with IFN-a (1000 IU/ml) for 30 min. The level of pY701-STAT1 was detected by immunoblotting with an anti–pY701-STAT1 Ab. (B) 293T cells transfected with either shCON or shHDAC3, together with FH-ATXN3, ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (C) Cells were transfected as in (A) and then were pretreated with IFN-a (60 IU/ml) for 20 h, followed by infection with VSV. The viral RNA levels of VSV in cells were analyzed by qPCR. (D) HepG2 cells transfected with either shCON or shHDAC3 were infected with VSV for 20 h, and viral RNA levels were analyzed by qPCR. Data are representative of three independent experiments (A) or from three experiments [(B–D) mean and SD of three independent replicates]. *p , 0.05, **p , 0.01, ***p , 0.001. n.s, not significant.

Article Snippet: Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from Addgene.

Techniques: Activity Assay, Transfection, Control, Western Blot, Luciferase, Infection

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, Ubiquitin Proteomics

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 Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Design and Screening of Engineered mi ATXN3 Constructs (A) Schematic representation of the human ATXN3 gene and mi ATXN3 binding sites. The ATXN3 gene (NG_008198.2) consists of 11 exons shown by the numbered black boxes. The white boxes represent the 5′ and 3′ UTRs. The CAG expansion in exon 10 is depicted by a red triangle. The position of the mi ATXN3 candidates are shown on top of the exons and indicated with numbers 1–11. mi ATXN3 _3, mi ATXN3 _8, and mi ATXN3 _9 are exon spanning. (B) Schematic representation of the mi ATXN3 constructs. Each construct was expressed by the CAG promotor, followed by the primary mi ATXN3 sequence in the miR-451 scaffold, and a human growth hormone polyadenylation (hGH poly(A)) signal. (C) Schematic representation of the Luc reporter. The whole sequence of the ATXN3 mRNA (NM_004993.5) was cloned downstream of the RL gene. In addition, FL was co-expressed from the vector as an internal control. (D) Dose-dependent knockdown of ATXN3 Luc reporter by mi ATXN3 constructs. HEK293T cells were co-transfected with 50 ng of the Luc reporter and 0.1, 1, 10, and 100 ng of the mi ATXN3 constructs. RL and FL were measured 2 days post-transfection, and RL was normalized to FL expression. Scrambled microRNA (miScr) served as a negative control and was set at 100%.

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Construct, Binding Assay, Sequencing, Clone Assay, Plasmid Preparation, Control, Knockdown, Transfection, Expressing, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Silencing of ATXN3 mRNA and Protein in HEK293T Cells (A) Endogenous knockdown ATXN3 mRNA by the selected mi ATXN3 candidates. qRT-PCR ATXN3 mRNA was performed on RNA from HEK293T cells that were transfected with 250 ng of mi ATXN3 _7, mi ATXN3 _9, or mi ATXN3 _11 for 3 days. mRNA input levels were normalized to GAPDH mRNA. Cells transfected with a GFP construct served as negative control, which was was set at 100%. (B) Silencing of total ataxin-3 protein. HEK293T cells were transfected as describe in (A), and protein expression was determined by western blot. α-Tubulin was included as internal control. Western blot intensity bands of ataxin-3 were quantitated and the knockdown was calculated relative to GFP. Data were analyzed using a multiple comparison one-way ANOVA to determine statistically significances of cells treated the mi ATXN3 constructs. The p values are listed in the graph as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Each graph represents the mean values with SD (n = 3).

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Construct, Negative Control, Expressing, Western Blot, Control, Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Silencing of ATXN3 mRNA by AAV5-miC in Human iPSC-Neurons (A) Dose-dependent silencing of ATXN3 in transduced iPSC-neurons. Frontal brain-like neurons were transduced with 2.4 × 10 12 , 2.4 × 10 11 , and 2.4 × 10 10 gc of mi ATXN3 _7, mi ATXN3 _9, or mi ATXN3 _11 incorporated into AAV5. RNA was isolated 2 weeks post-transduction and ATXN3 mRNA levels was determined by qRT-PCR. mRNA input was normalized to GAPDH and set relative to PBS-treated cells. (B) Levels of mature mi ATXN3 guide strands in transduced cells. Performed as described in (A). Expression of the mature mi ATXN3 _7, mi ATXN3 _9, and mi ATXN3 _11 was determined by small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to PBS-treated cells. (C) Relative mi ATXN3 expression levels in transduced cells by small RNA sequencing. Frontal brain-like neurons were transduced with 2.4 × 10 12 gc of mi ATXN3 _7, mi ATXN3 _9, or mi ATXN3 _11 incorporated in AAV5. Small RNA sequencing was performed 2 weeks post-transduction. The total amounts of small RNA reads corresponding to the three lead mi ATXN3 candidates are shown by the black arrows. The total amount of reads from other natural expressed endogenous microRNAs are shown in gray. (D) Processing of mi ATXN3 in iPSC-neurons by small RNA sequencing. Frontal brain-like neurons were transduced as described in (C). The secondary mi ATXN3 structure based on miRbase prediction is shown on the first row, including their predicted 22-nt guide strands shown in red. The sequence distributions of the different guide strand length (nt) mapping to mi ATXN3 _7, mi ATXN3 _9, and mi ATXN3 _11 pre-microRNA sequences in the miR451 scaffold were calculated as percentages (% reads).

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Transduction, Isolation, Quantitative RT-PCR, Expressing, RNA Sequencing, Sequencing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Vector Copy Distribution of AAV5 in SCA3 Knockin Mice (A) Schematic representation of the routes of administration. Three-month-old mice (n = 3) were injected i.c.v., or in the cisterna magna, or DCN with 2.43 × 10 13 gc/mL AAV5-mi ATXN3 _7, AAV5-mi ATXN3 _9, or AAV5-mi ATXN3 _11. 10 μL of AAV5 was injected either i.c.v. or in the cisterna magna, and 2 μL was injected bilaterally in the DCN. The injection sites are depicted in red. All mice were sacrificed 6 weeks after surgeries. (B–D) Vector copy distribution in cortex, cerebellum, and brain stem of mice injected i.c.v. (B), or in the cisterna magna (C) or DCN (D) with AAV5-miATXN3. DNA was isolated from the cortex, cerebellum, and brain stem tissues and qRT-PCR was performed to determine the vector copy distribution. The genome copies per μg of DNA were calculated for each brain region using a standard curve. No genomic copies were detected in untreated mice.

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Plasmid Preparation, Knock-In, Injection, Isolation, Quantitative RT-PCR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Silencing of Mutant Ataxin-3 in SCA3 Knockin Mice (A) Expression of mature mi ATXN3 guide strands in the cerebellum after DCN administration. Total RNA was isolated from the cerebellum for small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to AAV-GFP-treated mice. (B) Lowering of total ATXN3 mRNA in cerebellum of DCN-injected mice. Total RNA was isolated from cerebellum and qRT-PCR was performed to detect the mouse wild-type ATXN3 mRNA. RNA input levels were normalized to GAPDH and set relative to AAV-GFP treated mice. (C) Expression of mature mi ATXN3 guide strands in the brain stem after cisterna magna administration. Performed as described in (A). (D) Lowering of total ATXN3 mRNA in cerebellum of cisterna magna injected mice. Performed as described in (B). (E) Expression of mature mi ATXN3 guide strands in the brain stem after cisterna magna administration. Performed as described in (A). (F) Lowering of total ATXN3 mRNA in brain stem of cisterna magna-injected mice. Performed as described in (B). (G) Reduction of mutant ataxin-3 protein in the brain stem after cisterna magna delivery. TR-FRET immunoassay was performed on tissue homogenates to specifically detect the mutant ataxin-3 protein containing more than 37 glutamine repeats (no detection of wild-type mouse ataxin-3). (H) Reduction of mutant ataxin-3 protein in the cerebellum after cisterna magna delivery.

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Mutagenesis, Knock-In, Expressing, Isolation, Injection, Quantitative RT-PCR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Design and Screening of Engineered mi ATXN3 Constructs (A) Schematic representation of the human ATXN3 gene and mi ATXN3 binding sites. The ATXN3 gene (NG_008198.2) consists of 11 exons shown by the numbered black boxes. The white boxes represent the 5′ and 3′ UTRs. The CAG expansion in exon 10 is depicted by a red triangle. The position of the mi ATXN3 candidates are shown on top of the exons and indicated with numbers 1–11. mi ATXN3 _3, mi ATXN3 _8, and mi ATXN3 _9 are exon spanning. (B) Schematic representation of the mi ATXN3 constructs. Each construct was expressed by the CAG promotor, followed by the primary mi ATXN3 sequence in the miR-451 scaffold, and a human growth hormone polyadenylation (hGH poly(A)) signal. (C) Schematic representation of the Luc reporter. The whole sequence of the ATXN3 mRNA (NM_004993.5) was cloned downstream of the RL gene. In addition, FL was co-expressed from the vector as an internal control. (D) Dose-dependent knockdown of ATXN3 Luc reporter by mi ATXN3 constructs. HEK293T cells were co-transfected with 50 ng of the Luc reporter and 0.1, 1, 10, and 100 ng of the mi ATXN3 constructs. RL and FL were measured 2 days post-transfection, and RL was normalized to FL expression. Scrambled microRNA (miScr) served as a negative control and was set at 100%.

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Construct, Binding Assay, Sequencing, Clone Assay, Plasmid Preparation, Control, Knockdown, Transfection, Expressing, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Silencing of ATXN3 mRNA and Protein in HEK293T Cells (A) Endogenous knockdown ATXN3 mRNA by the selected mi ATXN3 candidates. qRT-PCR ATXN3 mRNA was performed on RNA from HEK293T cells that were transfected with 250 ng of mi ATXN3 _7, mi ATXN3 _9, or mi ATXN3 _11 for 3 days. mRNA input levels were normalized to GAPDH mRNA. Cells transfected with a GFP construct served as negative control, which was was set at 100%. (B) Silencing of total ataxin-3 protein. HEK293T cells were transfected as describe in (A), and protein expression was determined by western blot. α-Tubulin was included as internal control. Western blot intensity bands of ataxin-3 were quantitated and the knockdown was calculated relative to GFP. Data were analyzed using a multiple comparison one-way ANOVA to determine statistically significances of cells treated the mi ATXN3 constructs. The p values are listed in the graph as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Each graph represents the mean values with SD (n = 3).

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Construct, Negative Control, Expressing, Western Blot, Control, Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Silencing of ATXN3 mRNA by AAV5-miC in Human iPSC-Neurons (A) Dose-dependent silencing of ATXN3 in transduced iPSC-neurons. Frontal brain-like neurons were transduced with 2.4 × 10 12 , 2.4 × 10 11 , and 2.4 × 10 10 gc of mi ATXN3 _7, mi ATXN3 _9, or mi ATXN3 _11 incorporated into AAV5. RNA was isolated 2 weeks post-transduction and ATXN3 mRNA levels was determined by qRT-PCR. mRNA input was normalized to GAPDH and set relative to PBS-treated cells. (B) Levels of mature mi ATXN3 guide strands in transduced cells. Performed as described in (A). Expression of the mature mi ATXN3 _7, mi ATXN3 _9, and mi ATXN3 _11 was determined by small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to PBS-treated cells. (C) Relative mi ATXN3 expression levels in transduced cells by small RNA sequencing. Frontal brain-like neurons were transduced with 2.4 × 10 12 gc of mi ATXN3 _7, mi ATXN3 _9, or mi ATXN3 _11 incorporated in AAV5. Small RNA sequencing was performed 2 weeks post-transduction. The total amounts of small RNA reads corresponding to the three lead mi ATXN3 candidates are shown by the black arrows. The total amount of reads from other natural expressed endogenous microRNAs are shown in gray. (D) Processing of mi ATXN3 in iPSC-neurons by small RNA sequencing. Frontal brain-like neurons were transduced as described in (C). The secondary mi ATXN3 structure based on miRbase prediction is shown on the first row, including their predicted 22-nt guide strands shown in red. The sequence distributions of the different guide strand length (nt) mapping to mi ATXN3 _7, mi ATXN3 _9, and mi ATXN3 _11 pre-microRNA sequences in the miR451 scaffold were calculated as percentages (% reads).

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Transduction, Isolation, Quantitative RT-PCR, Expressing, RNA Sequencing, Sequencing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Vector Copy Distribution of AAV5 in SCA3 Knockin Mice (A) Schematic representation of the routes of administration. Three-month-old mice (n = 3) were injected i.c.v., or in the cisterna magna, or DCN with 2.43 × 10 13 gc/mL AAV5-mi ATXN3 _7, AAV5-mi ATXN3 _9, or AAV5-mi ATXN3 _11. 10 μL of AAV5 was injected either i.c.v. or in the cisterna magna, and 2 μL was injected bilaterally in the DCN. The injection sites are depicted in red. All mice were sacrificed 6 weeks after surgeries. (B–D) Vector copy distribution in cortex, cerebellum, and brain stem of mice injected i.c.v. (B), or in the cisterna magna (C) or DCN (D) with AAV5-miATXN3. DNA was isolated from the cortex, cerebellum, and brain stem tissues and qRT-PCR was performed to determine the vector copy distribution. The genome copies per μg of DNA were calculated for each brain region using a standard curve. No genomic copies were detected in untreated mice.

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Plasmid Preparation, Knock-In, Injection, Isolation, Quantitative RT-PCR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

doi: 10.1016/j.omtm.2019.10.008

Figure Lengend Snippet: Silencing of Mutant Ataxin-3 in SCA3 Knockin Mice (A) Expression of mature mi ATXN3 guide strands in the cerebellum after DCN administration. Total RNA was isolated from the cerebellum for small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to AAV-GFP-treated mice. (B) Lowering of total ATXN3 mRNA in cerebellum of DCN-injected mice. Total RNA was isolated from cerebellum and qRT-PCR was performed to detect the mouse wild-type ATXN3 mRNA. RNA input levels were normalized to GAPDH and set relative to AAV-GFP treated mice. (C) Expression of mature mi ATXN3 guide strands in the brain stem after cisterna magna administration. Performed as described in (A). (D) Lowering of total ATXN3 mRNA in cerebellum of cisterna magna injected mice. Performed as described in (B). (E) Expression of mature mi ATXN3 guide strands in the brain stem after cisterna magna administration. Performed as described in (A). (F) Lowering of total ATXN3 mRNA in brain stem of cisterna magna-injected mice. Performed as described in (B). (G) Reduction of mutant ataxin-3 protein in the brain stem after cisterna magna delivery. TR-FRET immunoassay was performed on tissue homogenates to specifically detect the mutant ataxin-3 protein containing more than 37 glutamine repeats (no detection of wild-type mouse ataxin-3). (H) Reduction of mutant ataxin-3 protein in the cerebellum after cisterna magna delivery.

Article Snippet: Real-time PCR amplification was performed with a customized TaqMan for human ATXN3 (assay ID Hs01026440_g1) or mouse ATXN3 (assay ID Mm00804702_m1) on an ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA, USA).

Techniques: Mutagenesis, Knock-In, Expressing, Isolation, Injection, Quantitative RT-PCR