PDIA3 is increased by METH in rodents <i>in vivo</i>.

<p>Time course of gene expression, determined by qRTPCR, in striatum from mice treated with 10 mg/kg METH at the indicated time points. (A) PDIA3 (B) HSPA5 (as a positive control). *p<0.05 as determined by a one-way ANOVA followed by a post hoc Tukey's test.</p

Increased PDIA3 suppresses ROS production.

<p>Cells treated with 500 µM METH for 1 h were assessed for production of ROS using DCFH-DA assay. A significant increase in ROS production is seen in cells knocked down for PDIA3, compared to PDIA3 expressors, both with and without METH treatment. Two-way ANOVA p<0.0001, Bonferroni post-tests. ***p<0.001. U – No treatment; M – METH treatment.</p

Primary rat striatal neurons stained for levels and distribution of ATP1A3 after 250 µM METH treatment for 24 h along with MAP2 staining for neuronal structure.

<p>(A) and with synaptic marker synaptophysin (B). DAPI was used to stain cell nucleus. Cells treated with METH show an increase and redistribution of ATP1A3 along the neuronal processes indicated by arrowheads and in the nerve terminals (inset). Scale bar = 10 µm.</p

Increased PDIA3 protects from METH toxicity.

<p>Examination of SK-N-BE(2) cells for expression and knockdown for PDIA3 by (A) western blot and (B) immunofluorescence. Scale bar = 10 µm. (C) Increased METH cytotoxicity in PDIA3 knockdown cells compared to the PDIA3 expressors. Cytotoxicity was assessed by a lactate dehyrogenase assay following 48 hrs exposure to 500 µM METH. ***p<0.001, unpaired Student's t-test.</p

METH induction of PDIA3 <i>in vitro</i> in rodent neurons.

<p>Primary rat striatal neurons (8 DIV) stained for levels and distribution of PDIA3 after 250 µM METH treatment for 24 h along with MAP2 staining for neuronal structure and DAPI for cell nucleus. Control cells show basal levels of PDIA3 while cells treated with METH show an increase and redistribution of PDIA3 along the neuronal processes indicated by arrowheads. Scale bar = 10 µm.</p

PDIA3 is increased in by METH in monkeys <i>in vivo</i>.

<p>Levels of PDIA3 mRNA expression from qRTPCR in brain regions from the two groups of animals, differing only by METH treatment. (A) caudate and (B) hippocampus. **p<0.01, unpaired Student's t-test.</p

List of genes up-regulated in both the caudate and hippocampus from SIV infected, METH and PBS administered monkeys.

<p>The gene symbol, gene name, p-value (unpaired Student's t-test) and fold change between the groups is indicated. Genes with p<0.01 and a fold change of >2 in both regions are shown.</p

Western blot analysis on rat striatal neurons pre-treated with indicated cytokines (10 ng each) for 6 h followed by METH treatment for 24 h showing increased ATP1A3 expression compared to METH only and cytokine only treatments.

<p>(M – METH, I – Interferon-γ, T – TNF-α). Bar graphs showing significant increase in ATP1A3 expression post treatment with the cytokines. Data represented as Mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus METH treatment.</p

Western blot analysis on rat striatal neurons pre-treated with ERK1/2 JNK and p38 inhibitors U1026, SP600125, and SB203580 respectively followed by METH treatment shows U1026 blocks the METH-induced increase in ATP1A3 expression.

<p>(A) but SP600125 or SB203580 do not (B, C). Data represented as Mean ± SEM of three independent experiments. **p<0.01 versus control, ##p<0.01 versus METH treatment.</p

Synaptic proteins differentially regulated between the two groups of monkeys.

<p>Unpaired student t-test was used to determine the significance.</p