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

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

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

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

Western blot analysis on rat striatal neurons pre-treated with the ERK inhibitor U1026 followed by cytokines treatment (6 h) in absence (A) and presence (B) of METH treatment for 15 min show abrogation of the increase in ATP1A3 expression. Data represented as Mean ± SEM of three independent experiments.

<p>*p<0.05, **p<0.01 versus control, #p<0.05 versus cytokine ± METH treatment.</p

Quantitative Proteomics by SWATH-MS Reveals Altered Expression of Nucleic Acid Binding and Regulatory Proteins in HIV-1-Infected Macrophages

Human immunodeficiency virus type 1 (HIV-1) infection remains a worldwide epidemic, and innovative therapies to combat the virus are needed. Developing a host-oriented antiviral strategy capable of targeting the biomolecules that are directly or indirectly required for viral replication may provide advantages over traditional virus-centric approaches. We used quantitative proteomics by SWATH-MS in conjunction with bioinformatic analyses to identify host proteins, with an emphasis on nucleic acid binding and regulatory proteins, which could serve as candidates in the development of host-oriented antiretroviral strategies. Using SWATH-MS, we identified and quantified the expression of 3608 proteins in uninfected and HIV-1-infected monocyte-derived macrophages. Of these 3608 proteins, 420 were significantly altered upon HIV-1 infection. Bioinformatic analyses revealed functional enrichment for RNA binding and processing as well as transcription regulation. Our findings highlight a novel subset of proteins and processes that are involved in the host response to HIV-1 infection. In addition, we provide an original and transparent methodology for the analysis of label-free quantitative proteomics data generated by SWATH-MS that can be readily adapted to other biological systems

MTT assay showing viability of cells post treatment with varying amounts of METH for 24 h.

<p>**p<0.01, ***p<0.001 (A). (B) Representative western blot confirming up regulation of ATP1A3 in rat striatal neurons treated with indicated concentrations of METH for 24 h as compared to controls. (C) Bar graphs showing significant increase in ATP1A3 expression post METH treatment versus control. Data represented as Mean ± SEM of three independent experiments. *p<0.05.</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