Plasmids FLQ10 and FLQ65, encoding N-terminal Flag and C-terminal myc tagged full-length ataxin-7 referred to as ATXN7Q10-Myc and ATXN7Q65-Myc have been previously reported . Plasmids encoding GFP-tagged WT, A4V or H48Q SOD1 constructs as well as RORalpha have been previously described [10, 48].
Cell culture and transfections
Generation of stable inducible PC12 cell lines expressing N-terminal FLAG- and C-terminal GFP-tagged ATXN7 with 10 (FLQ10 line) or 65 (FLQ65 line) glutamines have been described previously . In these cell lines expression of the corresponding proteins named ATXN7Q10-GFP and ATXN7Q65-GFP is induced upon removal of doxycline from the media. The FLQ10 and FLQ65 stable PC12 cell lines were grown at 37°C and 5% CO2, in DMEM (Invitrogen) supplemented with 10% horse serum (Invitrogen), 5% Tet System Approved fetal bovine serum (PAA), 100 μg/ml G418 (Invitrogen), 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulphate (Invitrogen), 100 μg/ml hygromycin (Invitrogen) and 1 μg/ml doxycycline (Sigma) when desired.
Human Embryonic Kidney 293 T (HEK 293 T) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (PEST, Invitrogen) at 37°C, 5% CO2.
For transient transfections, 7 × 105 HEK 293 T cells were seeded in 6 well plates and transfected 24 h later using Polyethylenimine (CellnTec) according to the product protocol.
Treatments and synthesis of gp91-TAT
Cells were treated with various concentration of H2O2 (0–0.5 mM) (Sigma), NAC (N-acetyl-L-cysteine) (0–5 mM) (Sigma) or BSO (buthionine sulfoximine) (0–2 mM) (Sigma). Apocynin, and α-tocopherol were used at a final concentration of 50 μM and 1 μM, respectively. The NOX complex inhibitor peptide gp91ds-TAT ([H]-R-K-K-R-R-Q-R-R-R-C-S-T-R-I-R-R-Q-L-NH2) and the control peptide Scramble-TAT ([H]-R-K-K-R-R-Q-R-R-R-C-L-R-I-T-R-Q-S-R-NH2) has been previously described  and were used at a concentration of 10 μM. The peptides were synthesized (SYRO multiple peptide synthesizer, MultiSynTech, Germany) on Fmoc-Rink- amide-chemmatrix resin (PCAS biomatrix inc.) using standard Fmoc solid-phase peptide synthesis. The peptide was cleaved using 95% TFA/2% water/2% triisopropylsilane/1% 1,2-ethanedithiol) for 3 h and precipitated in diethylether. The crude peptide was dried in vacuum overnight. The peptide was purified by HPLC on a Discovery® C-18 Supelco® column (Sigma-Aldrich, Sweden) using a gradient of acetonitrile/ water containing 0.1% TFA. Purity and identity was verified by analytical HPLC and by MALDI-TOF on a Voyager STR. The mass-spectrum was acquired in positive ion reflector mode using a-cyano-4-hydroxycinnamic acid as matrix (Sigma-Aldrich) (10 mg/ml, 7:3 acetonitrile: water, 0.1% TFA).
Cell lysis and Western blotting
Cell lysis and Western blotting was done as previously described . In brief, cells were lysed with RIPA buffer (Millipore) supplemented with protease inhibitors and the supernatant collected after centrifugation at 21,000 g at 4 °C for 10 min. Protein concentrations were determined with Bradford assay (Bio-Rad) and 10–20 μg of extract was subjected to SDS–PAGE. Proteins were transferred onto nitrocellulose membrane (Whatman), the membrane blocked and incubated with primary antibodies in 2% milk-TBST (100 mM Tris-buffered saline pH 7.4, 0.1% tween-20). Membranes were then washed, incubated with secondary antibody in 2% milk-TBST, and again washed with TBST. The protein of interest was visualized using SuperSignal West Pico chemiluminescent substrate or SuperSignal West extended duration substrate kits (Pierce) followed by film exposure or detection by a ChemiDoc XRS + imaging system (BioRad). Primary antibodies were used at the following concentrations; Ataxin-7  1:700, actin 1:500 (SC-1616, Santa Cruz), CAT 1:500 (SC-50508, Santa Cruz), SOD1 1:500 (SC-11407, Santa Cruz) and GSTA3 1:500 (gift from B. Mannervik). Signal intensities of target bands were quantified by Image lab software (BioRad). The relative intensity of the target protein in control and treated samples were acquired by first normalizing the target band with the corresponding actin intensity. The normalized intensity in control or treated samples was then divided by the sum of the normalized intensities of the target protein in control and all treated samples. The quote for the control sample was set to 100% and all treated samples in that experiment is shown as percent compared to control.
Filter trap assay
Filter trap assay was done as previously described . In short, cells were lysed in RIPA buffer and the pellets obtained after centrifugation at 21,000 g for 10 min were washed and resuspended in 50 μl DNAseI reaction buffer containing four unit of DNaseI enzyme (EN0521, Fermentas). The resuspended pellet, called the insoluble fraction, was incubated at 37 °C for 1 hr and Bradford assay (Bio-Rad) then used to determine the protein concentration in the sample. SDS and DTT were then added to a final concentration of 2% and 100 mM respectively before samples were heated at 95 °C for 5 min. Insoluble fractions were loaded and vacuum filtered through a 0.2 μm pore size membrane using a Bio-Rad dot-blot apparatus and a 0.1% SDS solution was added to each dot-blot slots twice to wash. The membrane was then removed from the dot-blot, blocked and subjected to immunoblotting using ATXN7 antibody as described above. Following immunoblotting, signal intensities of ATXN7 dots were quantified by Image lab software (BioRad) and normalized against the protein concentrations. For quantification of aggregation levels the average intensity from each dot was divided by the sum of the intensities of ATXN7 from all time points in that experiment and the intensity from the untreated control sample was set to 100%. All treated samples in that experiment is shown as percent compared to control.
Measuring of total ROS levels
FLQ10 or FLQ65 cells grown without Dox for 0, 2, 5, 8 or 11 days were seeded into 96-well culture plates in triplicate and grown in –Dox media for another 24 h before ROS was measured. Cells grown with doxycyline were used as control. ROS levels were measured by the oxidation-sensitive probe, dichloro-fluorescein-diacetate (DCHF-DA) (Sigma). Briefly, cells were washed two times with PBS after which PBS containing DCFH-DA (10 mM) was added to three wells with cells, whereas PBS only was added to three control wells. Fluorimetric measurement was taken immediately after addition for a period of 30 min using a flex station II plate reader. Cells treated with H2O2 (0.5 mM) were used as a positive control. The ROS levels were determined by subtracting the fluorescence of the PBS only treated well from the fluorescence of the DCFH-DA treated well to remove any potential background from GFP fluorescence.
Detection of mitochondria superoxide levels
FLQ65 cells grown without Dox for 5, 8 or 11 days were seeded into 96-well culture plates in triplicates and grown in –Dox media for another 24 h before mitochondria superoxide was measured. Cells grown with dox were used as control. Mitochondria superoxide levels were measured by the oxidative sensitive probe, MitoSOX™ Red reagent (Sigma). Briefly, cells were washed two times with PBS after which PBS containing MitoSOX™ Red reagent (5 μM) was added. Cells were incubated for 30 min after which fluoremetric measurement was taken for a period of 30 min using a flex station II plate reader. Cells treated with Antimycin A (100 nM) were used as a positive control.
Cell viability/toxicity measurements
WST-1 viability assays (Clontech) were performed according to the manufacturer’s protocol. Briefly, 50,000 induced or non-induced, treated or non-treated FLQ10 or FLQ65 PC12 cells were seeded in 96-well cell culture plate 24 h before viability measurements. Ten μl of WST-1 reagent was added to each well and after two hours of incubation the absorbance (450–690 nm) was measured on a Digiscan absorbance reader (Labvision). Following the WST-1 assay the protein concentration in each well was determined using Lowry assay (Bio-Rad). The obtained cell viabilities were normalized by protein concentration, and the value obtained from untreated non-induced sample was set to 100%.
Membrane integrity as a measure of toxicity was determined by analysis of lactate dehydrogenase (LDH) leakage from the cytosol of damaged cells using the CytoTox-ONE™ homogeneous membrane integrity assay (Promega), according to the manufacturer’s protocol. Briefly, 50,000 induced or non-induced cells were seeded in 12-well cell culture plate 24 h before analysis. Fifty μl of media from each sample/well was transferred to 96 well plates and incubated at room temperature for 20 min. A hundred μl of CytoTox-ONE (Promega) reagent was added to each sample and fluorescence measured using a flex station II plate reader at excitation wavelength of 560 nm and emission wavelength of 590 nm.
GSH level was measured using the GSH kit (Promega). Briefly, FLQ65 cells grown with or without Dox and treated with 50 μM Apo for 9 days were washed 2 times with PBS and lysed with GSH-Glo™ Reaction Buffer (Promega) for 30 min. Lysates were diluted 1:15 in deionized water and 10 μl of diluted lysate was transferred to 96-well plate in duplicates. Hundred μl of 1X GSH-Glo™ Reagent was added to each well and the samples incubate for 30 min at room temperature before 100 μl of prepared Luciferin Detection Reagent (Promega) was added to each well. After a 15 min incubation the luminescence was read using a microplate luminometer (Promega). The obtained luminescence value was normalized by protein concentration, and the value obtained from untreated non-induced sample was set to 100%.
Statistical analysis was done by one-way ANOVA followed by Tukey’s post-hoc test using Prism graph pad 5.0 or by two-tailed student t test. Data is represented as mean ± standard error of at least three independent experiments. In all cases, P <0.05 was considered to be statistically significant. Data are expressed as a percentage of control unless otherwise stated.