Abstract Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases. Keywords: Ataxin-1, Polyglutamine, Sleeping beauty transposon, Oxidative stress, Protein network, Ribosome Abbreviations: SCA1, Spinocerebellar ataxia type-1; polyQ, polyglutamine; IIBs, intranuclear inclusion bodies; SB, Sleeping Beauty; MSC, mesenchymal stem cell; FTIR, Fourier-transform infrared spectroscopy; AFM, atomic force microscopy; MS, mass spectrometry; PPI, protein-protein interaction; iPSC, induced pluripotent stem cell; NPC, neural progenitor cell; ROS, reactive oxygen species; DE, differentially expressed genes; GSEA, Gene Set Enrichment Analysis 1. Introduction Polyglutamine (polyQ) diseases are neurodegenerative disorders caused by trinucleotide (CAG) repeat expansions in the coding region of human genes, which result in abnormally large polyQ tracts in the produced proteins. In spinocerebellar ataxia type-1 (SCA1), the disease is caused by CAG expansions in the ATXN1 gene [[63]1]. The polyQ-expanded ataxin-1 (ATXN1) protein forms small oligomers and slowly aggregates into larger insoluble nuclear inclusions in the affected neurons [[64]2]. These are specifically detectable in the Purkinje cells of the cerebellum in SCA1 patients [[65]1]. Several lines of evidence suggest that the deposition of large inclusions may be protective due to the sequestration of smaller cytotoxic oligomers [[66]3]. However, recent findings indicate that insoluble inclusions might be also toxic, as they cause quiescence and activate necrotic mechanisms in cells [[67]4]. These events are thought to be induced by oxidative stress and the activation of checkpoint kinases which regulate the cell cycle [[68]5]. PolyQ aggregates have been extensively investigated in cell-free assays, using recombinant proteins produced in bacteria [[69][6], [70][7], [71][8]]. Studies in yeast, murine neurons and HeLa cells overexpressing fragments of polyQ-expanded genes provided valuable information on the structure and interactions of pathogenic inclusions in cellular systems [[72]9,[73]10]. However, a reliable aggregation model is still missing. While formation of insoluble inclusions was also observed in human induced pluripotent stem cell (iPSC)-derived neurons [[74]11], this phenotype cannot be easily reproduced [[75]12]. Therefore, novel protein aggregation models are needed that could be readily used to characterize polyQ inclusions generated in primary human cells. Human mesenchymal stem cells (MSCs) have been previously used for modeling of polyQ diseases [[76]13]. These cells have a partial ability to fuse with Purkinje neurons [[77]14], suggesting that they might contribute to the restoration of their physiological neuronal function. MSCs can be easily modified genetically using the Sleeping Beauty (SB) transposon system [[78]15], a safer alternative to viral approaches [[79]16]. Engineered cells proliferate and stably express human transgenes for several passages [[80][17], [81][18], [82][19]]. MSCs can also model replicative senescence, a cellular event that resembles aging and is associated with protein aggregation [[83]20]. Expression of aberrant, polyQ-expanded genes in human MSCs is expected to generate suitable cellular models supporting robust, measurable and reproducible pathogenic phenotypes. In this study, we report an inducible version of a previously described SB system [[84]21] that can be utilized for the controlled expression of pathogenic genes in cultured primary cells. We used this system to overexpress polyQ-expanded ATXN1 in human MSCs and observed that cells reproducibly accumulate intranuclear inclusion bodies (IIBs). First, the IIBs generate cellular oxidative stress and following prolonged induction become insoluble. Using a combination of biophysical methods [Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and mass spectrometry (MS)], we characterized their structure and protein composition. In addition, we analyzed transcriptional changes and mechanisms of dysfunction caused by the overexpression of mutant ATXN1 and the gradual accumulation of its protein product. We assessed the similarity of this model with human SCA1 cerebellum at the end-stage of the disease in terms of mechanisms leading to disease. In particular, our data indicate that aggregation of pathogenic ATXN1 perturbs the cellular protein synthesis machinery in a cerebellar protein-protein interaction (PPI) network. The potential implications of these results for the homeostasis of cerebellar neurons are discussed. 2. Materials and methods 2.1. Construction of Tet-On Sleeping Beauty transposon plasmids For the generation of a pT2-CMV/TetO[2]-EYFP-GW transposon plasmid, the CMV promoter was excised by HindIII-NheI digestion from a pT2-CMV-EYFP-GW plasmid [[85]21] and replaced by a CMV/TetO[2] promoter. ATXN1(Q30) (wild-type ATXN1; GeneID: 6310) or (Q82) cDNA was shuttled into pT2-CMV/TetO[2]-EYFP-GW plasmid by LR recombination, as previously described [[86]22]. Correct recombination was verified by BsrGI restriction digestion. An expression cassette encoding the SV40 promoter, β-globin intron, Tet-repressor (TetR) and bGH polyA signal flanked by SpeI restriction sites was synthesized and cloned into a pU57C plasmid. The cassette was subcloned by SpeI digestion into the MCS of a pT2-HB plasmid resulting in the generation of a pT2-TetR plasmid. Then, an expression cassette encoding the SV40 promoter, neomycin resistance gene (neo^R) and SV40 polyA site was excised by HindIII from a pT2-neo^R plasmid and inserted into the pT2-TetR plasmid, resulting in the generation of a bicistronic pT2-TetR-neo^R transposon. The orientation of TetR and neo^R expression cassettes was verified by EcoRV digestion. Plasmid isolation was performed using a DNA plasmid purification kit (Macherey-Nagel). 2.2. MSC and neural precursor cell (NPC) culture Isolation and characterization of human MSCs have been previously described [[87]17,[88]18]. Cells were cultured and expanded ex-vivo using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 IU/mL) and streptomycin (100 μg/mL). NPCs were obtained from healthy human iPSCs as previously described [[89]23]. Cells were seeded on Matrigel-coated plates and cultured in DMEM-F12/NeuroCult 1:1 supplemented with 1:200 N2 supplement, 1:100 B27 supplement lacking vitamin A, 3 μM CHIR 99021, 0.5 μM purmorphamine, 150 μM ascorbic acid (Sigma-Aldrich) and 1% penicillin/streptomycin/glutamine [[90]24]. 2.3. Generation of stable cell lines MSCs at passage 5 or NPCs at passage 9 were transfected with a mixture of SB100X/pT2 plasmids using Xfect reagent (Clontech), according to manufacturer's instructions. Briefly, 2 × 10^5 cells were seeded in a 6-well tissue culture plate. The next day, cells were transfected with 5 μg of total plasmid DNA encoding SB100X transposase and pT2 plasmids (1:9 ratio). The ratio of pT2-TetO[2]-YFP-ATXN1(Q30) or (Q82) and pT2-TetR-neo^R transposon plasmids was 5:1. For the generation of MSCs stably producing Venus, a pT2-GAGGS-Venus-neo^R transposon plasmid was used. Transfected cells were selected at day 7 post-transfection with 100 μg/mL G418 and expanded in the presence of the antibiotic. For transgene induction, fresh doxycycline (Dox) (2 μg/mL) was added every 48hrs in the culture medium. 2.4. Immunofluorescence Tet-On YFP-ATXN1(Q82) MSCs or NPCs were cultured in the presence or absence of doxycycline in CELLview glass bottom plates (Greiner Bio One). Then, cells were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.1% Triton-X 100 PBS before mounting with DAPI ProLong Gold antifade reagent (ThermoFisher). Cells were visualized in an Axiovert microscope equipped with an HBO 50 mercury lamp and reflectors with fluorescence filter sets. Image acquisition was performed with the Fluorescence Lite software module of AxioVision LE (Carl Zeiss). 2.5. Flow cytometry For quantification of transgene expression, Tet-On YFP-ATXN1(Q82) MSCs were measured in a Cytomics FC500 flow cytometer (Beckman Coulter) using the CXP2.2 software. For phenotypic characterization, 1 × 10^6 cells were stained with antibodies against hemopoietic (CD34, 45) or mesenchymal (CD29, 73, 90 and CD105) stem cell markers. Unstained cells or cells stained with IgG isotype antibodies were used as negative controls. 2.6. Immunoblotting MSCs (5 × 10^5) were lysed in 1% SDS PBS containing protease inhibitors (Calbiochem) and benzonase (Novagen). Cell extracts were analyzed in an 8% SDS-PAGE. Ectopic expression of YFP-ATXN1(Q30) or (Q82) was validated by Western blotting with the polyclonal anti-ATXN1 SA4645 antibody [[91]22] or anti-GFP antibody (MAB2510, Millipore). β-actin was detected with mouse monoclonal anti-β-actin antibody (sc-47778, Santa Cruz). After incubation with an appropriate alkaline-phosphatase conjugated secondary antibody, proteins were detected with NBT-BCIP (Applichem). 2.7. Filter retardation assay Insoluble protein inclusions were quantified using a filter retardation assay [[92]25]. Cell extracts of Tet-On YFP-ATXN1(Q30) or (Q82) MSCs (150 μg total protein) were mixed with equal volumes of 4% SDS and 100 mM DTT and heated at 95 °C for 5 min. Samples were diluted with 100 μl 0.2% SDS and filtered through a 0.2 μm cellulose acetate membrane. SDS-resistant inclusions retained on the membrane were detected using the polyclonal anti-ATXN1 SA4645 antibody (1:1000) [[93]22]. 2.8. FTIR spectroscopy Tet-On YFP-ATXN1(Q82) MSCs were cultured on MirrIR low-e-glass slides (Kevley Technologies, USA) washed with PBS and air dried. The spectra were taken using a Renishaw-Smiths micro Raman-FTIR microscope (Smiths-IlluminatIR, Smiths Detection, Hertfordshire, UK) and an ARO microscope lens (smithsARO 15x/N.A.88) at a range of 600–4000 cm^−1 wavenumbers with 100 measurements for the background spectrum and 300 for the sample spectrum with a 4 cm^−1 resolution. The low-e glass slide was placed on an automated holder and exposed to the instrument hood-controlled temperature and humidity environment of 22 °C and 37%, respectively. Background and sample absorption spectra were taken at a 100 μm diameter circular region. The sample absorption spectrum was calculated after background subtraction using the SynchronizIR software (Soft Imaging System GmbH, Germany). The second derivative spectra were obtained by a 5-point Savitzky-Golay algorithm of the smoothed spectrum (11-point binomial) using the GRAMS/32 software (Galactic Industries Corporation, Salem, NH, USA), according to relative processing [[94]26]. 2.9. Cytotoxicity assay Tet-On YFP-ATXN1(Q82) MSCs were cultured in 96-well tissue culture plates (2 × 10^4 per well). Equal volume of Caspase 3/7 Glo substrate (Promega) was added in each well and luminescence was measured in a Victor3 multilabel plate reader (PerkinElmer). Each experiment was performed in triplicate. 2.10. Detection of reactive oxygen species (ROS) 2′7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) (ThermoFischer) was used for detection of ROS. Tet-On YFP-ATXN1(Q82) were resuspended in PBS with or without the dye at a final concentration of 10 μM (loading buffer) and incubated at 37 °C for 30 min. The loading buffer was removed, cells were resuspended in prewarmed complete medium and further incubated at 37 °C for 5 min. The absorption and emission of the oxidation product were measured at 493 and 520 nm, respectively. Each experiment was performed in triplicate. 2.11. Proteasome peptidase assay Chymotrypsin-like (CT-L) proteasome activity was assayed in protein extracts with the hydrolysis of a specific fluorogenic peptide, namely LLVY-AMC, at 37 °C for 30 min, as previously described [[95]27]. Proteasome activity was determined as the difference between the total activity in protein extracts and the remaining activity in the presence of 20 μM MG132. Fluorescence was measured using a VersaFluor fluorescence spectrophotometer (Bio-Rad). Protein concentrations were determined using the Bradford method with bovine serum albumin as standard. 2.12. Preparation of samples for AFM Cells were either kept in culture medium or fixed with 4% PFA PBS before observation. For imaging of nuclei, Tet-On YFP-ATXN1(Q82) MSCs were treated as previously described [[96]28]. In brief, AFM images of nuclei were obtained by cell lysis in 0.25% NP-40 hypotonic Tris-HCl pH 7.4 buffer followed by fixation. For observation of polyQ inclusions, cells were lysed in 0.1% SDS PBS and purified inclusions attached to the plastic surface were immersed in PBS. 2.13. AFM imaging and mechanical characterization of nuclear content Hybrid mode imaging, contact mode and Young's modulus mapping were performed using either Bruker Dimension FastScan (Bruker Nano Surfaces) AFM or JPK NanoWizard 3 (JPK). Bruker SCANASYST-FLUID+ and SNL-10 A and B silicon nitride cantilevers equipped with silicon tip (both from Bruker Nano Surfaces) were used for imaging. Sensitivity and cantilever spring constant were calibrated by the routine procedure recommended by the instrument producer. Hybrid mode imaging (Bruker QNM mode) with SCANASYST-FLUID + probe was employed to record semi-quantitative images of isolated nucleus in PBS buffer. Images covering an area of 15 × 15 μm^2 and 2.5 × 2.5 μm^2 area (1500 × 1500 px^2 resolution) were recorded with a set point of 0.75 nN and lifting height of 150 nm, (Z-piezo range 0.75 μm and 0.35 μm, respectively). Images covering the whole nucleus (20 × 20 μm^2, 1024 × 1024 px^2 resolution) as well as the detailed view of 5 × 5 and 2 × 2 μm^2 were recorded by JPK Quantitative Imaging mode (hybrid mode) with a set point of 0.35 nN and lifting height of 90 nm (Z-piezo range was shortened to 3.0 μm). Cells fixed on a standard microscopic glass slide were visualized by constant force contact mode (JPK NanoWizard 3), when the Bruker SNL-10A cantilever was used. Set-point equal to 5.0 nN, feedback setting iGain = 50.0 Hz and PGain = 0.001 were the operating parameters for capturing images of 50 × 50, 25 × 25 and 10 × 10 μm^2 (2048 × 2048 px^2). For mapping of Young's modulus of living cells a previously published procedure [[97]29,[98]30] was adopted. Briefly, Bruker SNL-10B probe was used to record 64 × 64 maps of force-distance curves (set-point 1 nN, Z-length 15 μm, time per curve 0.5 s). The recorded force-distance (FD) curves were fitted with the Bilodeau modification of the Hertzian model. Final post-processing and editing of the images and stiffness maps was performed using the Gwyddion software ver. 2.44 [[99]31]. 2.14. Force-distance curves post-processing Force mapping provides a network of force-distance curves, measuring the dependence of the tip-sample interaction force on the tip height above the surface. The absolute value of Young's modulus can be determined by fitting the FD curve to the modified Hertzian-Sneddon equation: [MATH: F(δ)=2Et< mi>anαπ(1−υ2)δ :MATH] where F is the measured force, E is the Young's modulus, ν is the Poisson's ratio (0.5 for incompressible materials), δ is the tip-sample separation (obtained by correction of the cantilever height to its bending) and α is the half-angle to face of pyramidal tip (reflects the tip geometry). The data processing module of the JPK software was used to process the maps of FD curves. The resulting Young's modulus maps were exported in order to be post-processed using the Gwyddion software. Data masking and final export of stiffness maps were performed also using the Gwyddion software. 2.15. Mass spectrometry For proteomic analysis of insoluble polyQ inclusions, Tet-On YFP-ATXN1(Q82) MSCs at D10 were lysed in 0.1% SDS PBS. Intact fluorescent polyQ inclusions were sorted from cell extracts in a BD FACSAria IIu instrument (BD Biosciences) after doublet exclusion and using an unstained control sample to exclude possible autofluorescence. Protein samples were processed by the filter-aided sample preparation (FASP) method [[100]32]. Proteins were alkylated, digested by trypsin on filter unit membrane and resulting peptides were eluted by ammonium bicarbonate buffer. The peptide mixture was dried under vacuum and transferred using peptide extraction procedure to TPX vial. LC-MS/MS analysis was done using the RSLCnano system (Thermo Fisher Scientific) on-line connected to Impact II Ultra-High Resolution Qq-Time-Of-Flight mass spectrometer (Bruker). The analytical column outlet was directly linked to the CaptiveSpray nanoBooster ion source (Bruker ). MS and MS/MS spectra were acquired in a data-dependent strategy with 3 s long cycle time. For quantitative mass spectrometry, technical triplicates of Tet-On YFP-ATXN1(Q82) MSCs at D10 or D0 were lysed in SDT buffer. LC-MS/MS analysis of peptide mixture was done using RSLCnano system (Thermo Fisher Scientific) on-line connected to Orbitrap Q-Exactive HF X system (Thermo Fisher Scientific). The analytical column outlet was directly linked to the Digital PicoView 550 (New Objective) ion source. ABIRD (Active Background Ion Reduction Device, ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy selecting up to top 20 precursors. The analysis of the mass spectrometric data was carried out using Maxquant (version 1.6.3.3) with Andromeda search engine utilization. Search was done against the UniprotKB Human (version 20180912) and Maxquant's contamination databases (downloaded with given version). Mass tolerances for peptides and MS/MS fragments were 4.5–10 ppm and 0.05 Da, respectively. Oxidation of methionine, deamidation (N, Q) and protein N-terminal acetylation as optional modifications, carbamidomethylation (C) as fixed modification and two enzyme miss cleavages were set for search. Peptides and proteins with false discovery rate (FDR; q-value) < 1% were considered. Maxquant label free quantification algorithm (MaxLFQ) was applied for global data normalization [[101]33] (minimal ratio count 1). MQ protein group list was processed via KNIME Analytics Platform (v. 3.7.1). Results have been deposited in the PRIDE Archive ([102]https://www.ebi.ac.uk/pride/archive) under accession number PXD012709. 2.16. RNA-sequencing Total RNA was extracted from Tet-On YFP-ATXN1(Q82) MSCs using the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN), according to manufacturer's instructions. For library preparation, mRNA was enriched from 250 ng total RNA using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), according to manufacturer's protocol. Sequencing libraries were prepared from mRNA using SENSE Total RNA-seq Library Prep Kit (Lexogen), according to manufacturer's protocol. Single-end 75 bp sequencing was performed in a NextSeq 500 instrument (Illumina). In average, more than 30 million reads per time-point were obtained. Post-mortem human cerebellum from a 74-year-old female SCA1 patient and an age-/sex-matched healthy individual (Sample ID: A136/02 and A158/14, respectively) were obtained from the MRC London Neurodegenerative Diseases Brain Bank. Total RNA was extracted from frozen samples using the Direct-zol RNA Kit (Zymo Research). An aliquot of total RNA was loaded on Total RNA Pico Chip and measured for its integrity using Bionalyzer 2100 (Agilent Technologies). Sequencing libraries were prepared using KAPA RNA HyperPrep with Ryboerase kit, according to manufacturer's instructions (Roche). High throughput single-indexed, paired-end 75 bp sequencing was performed in a HiSeq 4000 instrument (Illumina). RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI ([103]www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-7713 and E-MTAB-7714. 2.17. Data analysis and visualization of RNA-seq data The quality control was performed based on the FastQC software (Babraham Institute, Cambridge, UK). Samples with a low number of raw reads (less than 10 million per sample) were excluded from downstream analysis. Reads were trimmed and filtered using Trim Galore! and consequently aligned against the UCSC hg19 reference genome using the HΙSAT2 protocol [[104]34]. Overall alignment rate for all samples ranged between 89% and 93%. Transcriptome assembly was performed using StringTie and differential expression calculation was performed using Ballgown. Gene expression levels were normalized to Fragments Per Kilobase of transcript per Million (FPKM). Subsequent data analysis was carried out in the R environment. T-test was applied to compare FPKM levels between the two different groups. Hierarchical clustering analysis was performed applying the R package “gplots” and FPKMs were normalized to z-score. The agglomerative hierarchical clustering was performed using the Euclidean distance metric and the complete agglomeration method. To compute the principal components (PCs), the prcomp function was applied. Gene set enrichment analysis (GSEA) was calculated using the clusterProfiler R package [[105]35]. Gene Ontology (GO) and Pathway enrichment analyses were performed using the online tool Enrichr where KEGG and PANTHER pathways were examined [[106]36]. Venny 2.1 (BioinfoGP, CNB–CSIC, [107]http://bioinfogp.cnb.csic.es/tools/venny/index.html) was utilized to overlap the differentially expressed genes from different comparisons. 2.18. Construction of a PPI network The PPI network in which the products of differentially expressed (DE) genes participate was constructed using release 2 of the Human Integrated Protein-Protein Interaction rEference (HIPPIE) [[108]37]. Only cerebellum-specific interactions with HIPPIE confidence scores of at least 0.63 (median of all scores in the database) were considered, which resulted in a network of 516 proteins. 2.19. Statistical analysis Statistical analysis was performed using the GraphPad Prism software (San Diego, USA). All cell-based assays were performed in triplicates and results are shown as mean ± SEM calculated by a T-test. For AFM measurement of cell stiffness, a one-way ANOVA with Bonferroni post-hoc test was used. To determine if the size of the largest connected component (LCC) of the constructed PPI network (328 proteins) was greater than expected by chance, it was compared with a distribution of LCC sizes using a z-test. This distribution resulted from randomly sampling 516 proteins 1,000 times from the complete cerebellum-specific PPI network. A z-test was also employed to determine if the number of DE genes, common to cells and the SCA1 patient and also present in the PPI network, was significantly larger than expected by chance. Specifically, we randomly sampled 185 genes from the 3,923 SCA1-differentially-expressed pool 1,000 times and intersected the sample with the 328 members of the PPI LCC. Then, we checked whether 109 (the actual size of the intersection between the 185 SCA1-DE genes and the LCC) was significantly greater than the constructed random distribution. Differences in protein levels of the network were investigated by LIMMA test. P-values were adjusted with the Benjamini-Hochberg method. 3. Results 3.1. Inducible Sleeping Beauty transposon system to express ATXN1 variants In order to develop a stable ATXN1 expression model, an inducible Tet-On SB transposon system was generated. The system consists of two pT2-based expression plasmids, a Tet-On YFP-tagged Gateway destination plasmid, in which ATXN1(Q82) was shuttled [resulting in YFP-ATXN1(Q82)], and a TetR-neo^R bicistronic transposon plasmid encoding for the TetR protein and the neomycin resistance gene ([109]Fig. 1A–B). Using the hyperactive SB100X transposase [[110]15], a stable expression cell line was generated in human MSCs. Fig. 1. [111]Fig. 1 [112]Open in a new tab Generation of Tet-On YFP-ATXN1(Q82) MSCs using a novel inducible SB transposon system. (A) Schematic overview of the Tet-On principle. In the absence of tetracycline, TetR protein dimers bind to the CMV/TetO[2]promoter and suppress transgene expression. In the presence of tetracycline, TetR adopts a different conformation and loses its affinity for the CMV/TetO[2] promoter allowing transgene expression. (B) Components of the Tet-On SB transposon system. The system is comprised of two pT2-based plasmids, a Tet-On Gateway compatible and a bicistronic TetR-neo^R transposon. (C) Fluorescence microscopy of Tet-On YFP-ATXN1(Q82) MSCs in the absence (D0, left) or presence (D1, right) of Dox (scale bar = 25 μm). (D) Flow cytometry (histogram plot) of uninduced (blue color) and induced (green color) cells. (E) Immunoblot detection of transgene expression in extracts of uninduced and induced cells using anti-ATXN1 or anti-GFP antibodies. β-actin was used as a loading control. . (For interpretation of the references to