Effect of 6-OHDA treatment for 24 hrs on CAD cells.

<p>40 µg of cell lysate was resolved by SDS-PAGE, transferred to nitrocellulose membrane and stained for TID1, phosphorylated ERK, total ERK, caspase 3 and actin as loading control.</p

TID1 is widely expressed in brains of 6-OHDA-lesioned rats.

<p>TID1 was widely detected by immunohistochemistry in frozen rat brain serial sections. (<b>A</b>) Hippocampus: non-lesion hemisphere. (<b>B</b>) Hippocampus: lesion hemisphere. (<b>C</b>) Substantia nigra: non-lesion hemisphere. (<b>D</b>) Substantia nigra: lesion hemisphere. (<b>E</b>) Striatum: non-lesion hemisphere. (<b>F</b>) Striatum: lesion hemisphere. Scale: 100 µm.</p

6-OHDA treatment causes ERK phosphorylation and does not activate the Caspase pathway.

<p>27–33 days following left hemisphere injection of 6-OHDA, brain regions were dissected. 30 µg of homogenate isolated form the striatum (STR), midbrain (MB) and hippocampus (HPC) were heated at 95°C for 5 min., resolved by SDS-PAGE, transferred to nitrocellulose membrane and probed for TID1, phosphorylated ERK (phosphorylated p44/42 MAPK), total ERK expression (total p44/42 MAPK) and caspase 3. Lanes from left to right are: control (rat 18), 6-OHDA rats 1, 2, 3, 6, 7, 12, 13, and 16.</p

Unilateral 6-OHDA-lesions increased TID1 (DnaJA3) chaperone breakdown.

<p>(<b>A</b>) Following left hemisphere injection of 2 µl of 4 mg/ml 6-OHDA or saline, rats were tested for neurodegeneration and the indicated brain region were dissected 27–33 days following injection. 30 µg of solubilized midbrain was heated at 95°C for 5 min, resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-TID1 monoclonal antibody. The Western blot shown is representative of 11 6-OHDA/saline pairs of rats. Actin is shown as a loading control. (<b>B</b>) Midbrain samples were fractionated into soluble and insoluble and subjected to Western analysis. (<b>C</b>) TID1 expression in the indicated regions of saline injected rats was evaluated by Western analysis with anti-TID1 monoclonal antibody and quantitated by Quantity One (BioRad). <b>(D)</b> TID1 expression in 30 µg of solubilized hippocampus. The Western blot shown is representative of 11 6-OHDA/saline pairs of rats. Actin is shown as a loading control.</p

The performance of rats is influenced by 6-OHDA-lesions.

<p>(<b>A</b>) Sensory Impairment test, (<b>B</b>) Gait Symmetry Impairment test (<b>C</b>) Food Handling Impairment test in 6-OHDA lesion and saline control rats. Following left hemisphere injection of 2 µl of 6-OHDA or saline, rats were tested for behavioral deficits. A higher behavioral score indicates greater behavioral deficit. *** indicates significance p<0.001.</p

Primers used for qRT-PCR gene expression profile validation.

<p>Primers used for qRT-PCR gene expression profile validation.</p

Global gene expression analysis by microarrays was validated by RT-PCR analysis of selected genes.

<p><b>A, </b><i>Lgals3</i>; <b>B, </b><i>S100a4</i>; <b>C, </b><i>Vim</i>; <b>D, </b><i>B2m</i>; <b>E, </b><i>Egr1</i>; <b>F, </b><i>F2r</i>; <b>G, </b><i>Fabp7</i>; <b>H, </b><i>Gfap</i>; <b>I, </b><i>Hmgn.</i> All measurements were compared to naïve controls values. <i>Gadph</i> was used as a reference control for calculation of gene expression ratio. PCR-product amplification was confirmed by agarose electrophoresis gels (photographs).</p

Cumulative effects of prenatal and adult stress elevated HPA axis activity.

<p><b>A,</b> Adrenal gland weight; <b>B,</b> Plasma CORT levels. Animals stressed both prenatally and in adulthood showed larger adrenal glands and elevated CORT levels in response to the ischemic lesion. *p≤0.05.</p

Tactile stimulation restored forelimb function after ischemic lesion.

<p><b>A,</b> Success rate in the single pellet reaching task; <b>B,</b> Number of pellets obtained; <b>C,</b> Sequence of video frames depicting grasp, supination 1 and 2, and release components; <b>D,</b> Qualitative assessment of reaching components. Note that the ischemic lesion reduced quantitative (success, pellets obtained) and qualitative aspects of skilled reaching. Cumulative effects of prenatal and adult stress further reduced skilled reaching ability. TS promoted reaching success and movement ability. *p<0.05, **p<0.01.</p

Experimental design.

<p>Time-course of procedures. Adult male offspring was pre-trained and tested in a pellet reaching task and skilled walking. Blood samples were collected three times for corticosterone assessments. Stress and TS were induced for four weeks. Transcriptomic analyses were performed after behavioural testing was completed.</p