The Journal of Neuroscience, September 27, 2006, ():
Cytosolic Catechols Inhibit 
-Synuclein Aggregation and Facilitate the Formation of Intracellular Soluble Oligomeric Intermediates
J. Neurosci. Mazzulli et al.
26: 10068
Supplemental data
Files in this Data Supplement:
- supplemental material
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Figure S1. Alpha-Synuclein expression analysis in stably transfected SH-SY5Y cells. SH-SY5Y cells were stably transfected with empty vector plasmid, wild-type or A53T alpha-syn expressing plasmids. Cells were cultured with DMSO (vehicle) or retinoic acid for 5 days and Triton-soluble lysates were analyzed for alpha-syn expression levels by Western blot analysis using the monoclonal antibody Syn 211. Representative blots are shown in figure 1a. Intensities of alpha-syn Western blot bands were quantified by densitometry and normalized to NSE using fluorescent-labeled secondary antibodies (see Methods). Alpha-syn expression levels are increased in SH-SY5Y cells as a result of differentiation. Syn/NSE ratios with DMSO or RA respectively: Vector=0.1±.03, 0.2±0.004; wt alpha-syn=15±0.5, 26±0.7; A53T alpha-syn= 9±0.03, 12±0.3. Values are the mean ± sem, n=3, *p<0.05.
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Figure S2. Qualitative analysis of aggregates in A53T alpha-syn SH-SH5Y cells infected with lentivirus-containing empty vector or TH expression plasmids. A53T alpha-syn expressing cells were treated as described in figure 3 and analyzed for the number of cells containing a) large juxtanuclear (JN) aggregates (n=4, *p<0.001; ‡p=0.04) b) thioflavin S-positive signals (Thio S) (n=3, *p<0.001; ‡p=0.05) or c) TH positive cells containing large JN aggregates (n=3). Representative large JN aggregates are shown in figures 1c, bottom row and 3a vector condition. Only cells containing thio S positive structures that co-localized with alpha-syn puncta were counted as positive. Analysis of only TH-positive cells reveals that TH RR-EE infected cells are devoid of large JN aggregates (ND = not detectable). Values are the mean ± sem.
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Figure S3. Pharmacological manipulation of catechols in TH RR-EE infected A53T alpha-syn cells reveals that alpha-syn aggregation is modulated by dopamine. A53T alpha-syn cells were pretreated for 24 hours with either 2μM HCl (Vehicle), 0.5mM alpha-methyl-p-tyrosine (alpha-MT), or 0.2mM NSD 1015 (NSD), then infected with lentivirus-containing TH RR-EE expression plasmids and differentiated for 5 days with RA. a) Quantification of intracellular catechol levels by HPLC-ECD demonstrates effective inhibition of catechol synthesizing enzymes. Values are the mean, ± sem, n=3 b) Gel filtration/Western blot analysis of Triton-soluble fractions using Syn 211. c) Densitometric quantification of Triton-insoluble alpha-syn using LB509 and Alexa 680-conjugated fluorescent secondary antibodies (n=2).
