wig-1 knockdown does not affect p53 mRNA or protein levels following wig-1 ASO treatment in mice.

<p><i>A) mRNA and protein were extracted from BACHD</i> striatum of mice treated with 75 µg of wig-1 ASO, PBS, or control ASO. B) p53 mRNA and protein levels in mouse FVB striatum following ASO injection with increasing doses of wig-1 ASO, and C) Effects of wig-1 ASO treatment on p53 mRNA and protein levels in mouse liver. Data are expressed as means ± SEM (<i>n</i> = 4).</p

Genes up-regulated in BACHD mouse brain following wig-1 ASO treatment.

<p>Ingenuity™ Pathway Analysis was used to ascribe up-regulated genes with potential functions (additional genes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029429#pone.0029429.s003" target="_blank">Figure S3</a>).</p

Real Time-PCR confirmation of down-regulated mRNAs following wig-1 ASO treatment in mice.

<p>mRNA levels of <i>Auts2, Robo2, Plekha5, and IMMP2L</i> in (A) BACHD striatum, (B) BALB/c liver, or (C) FVB striatum was compared to control animals. Data are expressed as means ± SEM (<i>n</i> = 4; *<i>p≤0.1</i>; **<i>p≤0.05;</i> ***<i>p≤0.01</i>).</p

Effects of wig-1 ASO intrastriatal treatment on wig-1 and HTT levels in BACHD striatum.

<p><i>5-month-old</i> male <i>BACHD</i> mice received 75 µg single bolus injection of wig-1 ASO, PBS, or control ASO in striatum. Animals were sacrificed after one, or three weeks. A) Striatum was harvested and total RNA was prepared from these sections, and used for real-time quantitative RT-PCR analysis to evaluate the expression of wig-1 mRNA. B) Tissue homogenates were prepared from striatum and used for analysis of Wig-1 and HTT protein levels. Data are expressed as means ± SEM (<i>n</i> = 4; **<i>p≤0.05</i>; ***<i>p≤0.01</i>). Letters A1–A4, B1–B4, D1–D4 refer to individual animals.</p

Mouse Wig-1 ASOs specifically reduce wig-1 mRNA levels in vitro and in vivo.

<p>A) oligonucleotides were 20 nucleotides in length and chemically modified with phosphorothioate in the backbone and 2′-<i>O</i>-methoxyethyl (MOE) on the wings with a central deoxy gap (“5-10-5” design). B) b.END cells were transfected with indicated concentration of wig-1 ASOs. RNA was extracted 24 hours after transfection and analyzed by RT-PCR to determine wig-1 mRNA levels. C) Male <i>BALB/c</i> mice were injected intraperitoneally with 4 different wig-1 ASOs at 100 mg/kg body weight per week or with saline for 4 weeks. Total RNA was prepared from liver, and used for real-time quantitative RT-PCR analysis to evaluate wig-1 mRNA levels. Data are expressed as means ± SEM (<i>n</i> = 4, ***p≤<i>0.01</i>).</p

Up-regulation of Gpnmb mRNA levels following wig-1 ASO treatment.

<p>A) Gpnmb mRNA levels in BACHD striatum; B) FVB striatum, and C) BALB/c liver. Data are expressed as means ± SEM (<i>n</i> = 4; *p≤0.1; **<i>p≤0.05</i>).</p

Heatmap of the <i>log2</i> normalized intensities of the significantly differentially expressed 260 gene probes on the array.

<p>The columns represent the PBS, ASO treated, and negative control samples and the rows represent the 260 differentially expressed gene probes on the array. The heatmap shows that the majority of the differentially expressed genes show mostly upregulation after ASO treatment. This is shown by the transition of probe intensities to higher values relative to the PBS and negative control samples for the 260 gene probes. The dendrogram on the left of the heatmap shows the clustering of genes having similar profiles across samples. The dendrogram on top of the heatmap shows the clustering of the samples. The PBS and negative control samples clustered together whereas the ASO treated samples clustered separately.</p

Genes down-regulated in BACHD mouse brain following wig-1 ASO treatment.

<p>Ingenuity™ Pathway Analysis was used to ascribe down-regulated genes with potential functions (additional genes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029429#pone.0029429.s002" target="_blank">Figure S2</a>).</p

Effects of wig-1 ASO treatment on PKCε levels.

<p>PKCε mRNA and protein levels in BACHD and FVB striatum, and BALB/c liver were determined in animals treated with wig-1 ASO and compared to control animals. A) Effects of wig-1 ASO treatment on PKCε <i>mRNA levels. </i><i>B</i>) Effects of wig-1 ASO treatment on PKCε <i>protein levels</i>. Data are expressed as means ± SEM (<i>n</i> = 4; **<i>p≤0.05</i>; ***<i>p≤0.01</i>).</p

Inclusion appearance in various cortical and striatal regions in zQ175 heterozygous mice.

<p>(<b>A</b>) Using an automated microscope, whole mouse brain sections were scanned by high resolution multi-image acquisition. Individual images were assigned to distinct areas within the cortex including the cingulate cortex (ccx) and motor cortex (mcx), or within the striatum, including dorsal (d)/ventral (v) and medial (m)/lateral (l) parts to allow region-specific automated multiparametric analysis. (<b>B</b>) Region-specific analysis in the striatum of nuclear mHTT inclusions in MSNs. Inclusion number was found to be significantly higher in lateral quadrants (ld and lv) than in medial ventral quadrant at 8 and 12 months old zQ175 mice. (<b>C</b>, <b>D</b>) Subregion specific analysis in the cortex showing quantification of the number of nuclear (<b>C</b>) and extranuclear (<b>D</b>) mHTT inclusions in the cingulate and motor cortex over time. A significantly higher number of inclusions were detected in the ccx compared to mcx region in zQ175 heterozygous mice at 12 months of age. Data are displayed as mean +/-SD. Statistical analysis was performed by two-way ANOVA and Sidak’s multiple comparisons’ test. Mean values were calculated for every age and region using an n of 8 animals with 6 sections per animal; *p<0.05; **p<0.01; ***p<0.001.</p