The Journal of Neuroscience, January 24, 2007, ():
The Induction Levels of Heat Shock Protein 70 Differentiate the Vulnerabilities to Mutant Huntingtin among Neuronal Subtypes
J. Neurosci. Tagawa et al.
27: 868
Supplemental Data
Files in this Data Supplement:
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Supplementary Figure 1
Expression levels of polyQ proteins were equivalent in different types of neurons.
To examine whether each polyQ protein is expressed at a similar level in different neurons, we compared the expression among three types of neurons.
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Supplementary Figure 2
Different polyQ proteins were expressed at equivalent levels in primary neurons.
To analyze the efficiencies of different adenovirus vectors, expression levels of different polyQ proteins in cortical neurons were detected with different antibodies. The same filter was reprobed with three different antibodies that detect different aggregation states of polyQ proteins. After collecting all the blots, expression levels of the different polyQ proteins were found to be almost similar.
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Supplementary Figure 3
Western blot with subcellular fractions confirmed that HSF1 was not translocated from the cytoplasm to the nucleus, indicating that HSF1 is not activated under the expression of mutant htt.
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Supplementary Figure 4
The relationship between aggregation patterns of mutant htt and the expression levels of p53 in Hela cells
N-18, which is suitable for detection of htt, aggregates in immunocytochemistry, delineates two types of aggregates namely, cytoplasmic dot-like aggregates at centrosome (aggresome), and peri-nuclear ring-like aggregates (ring), as described previously (Tagawa et al., 2004) (upper panels). Signal intensities of p53 were collected in two types of aggregate-positive cells (arrows) and aggregate-negative cells (arrowheads) by using AQUACOSMOS (Hamamatsu Photonics. The lower panel shows the mean +/- SD of p53 signals in each type of cells. Only a weak difference was detected between aggregate-negative cells and ring-like aggregate-positive cells (p<0.05, Student’s t-test).
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Supplementary Figure 5
CBF and p53 regulate the expression of hsp70 in Hela cells in positive and negative manners, respectively.
(A) The construction of reporter plasmids used for CAT assays.
(B) CAT assays with the above mentioned reporter plasmids in Hela cells. The left two lanes are negative and positive controls. The reporter plasmids were constructed by subcloning the upstream sequence into p0CAT. The basal expression of pHsp70CAT is remarkably higher than that of p0CAT, which is very low (lane 3). When both of the CCAAT boxes are deleted, CAT activity is drastically decreased (lane 4). Deletion of either of the two CCAAT boxes reduces the CAT activity only slightly (lane 5, 6). Lane 3-6 show the CAT activities of Hela cells infected by empty adenovirus AxCA, and lane 7-10 are those infected by AxCA-htt(exon1)111Q. The expression of mutant htt remarkably increased CAT activity (lane 3 vs. lane 7), whereas it was repressed when both of the CCAAT boxes were deleted (lane 8), indicating that the induction of hsp70 by mutant htt, is mediated by the cooperation of the two CCAAT boxes. The difference in CAT activities among lane 7-10 is clear from non-acetylated cloramphenicol (arrow). A quantitative analysis of CAT activities (n=6, boxed) in lanes 3-10 is shown in the lower graph.
(C) CBF transactivates hsp70 promoter activity, while co-expression of p53 repressed it either in the absence of (lane 1-3) or in the presence of mutant htt (lane 4-6). The lower graph shows a quantitative analysis of the CAT activities (boxed).
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Supplementary Figure 6
The response of p53 to bleomycin is different in cerebellar and cortical neurons.
To induce p53, 30μg/ml of bleomycin was added to the primary culture. The foci formation of p53 is more visible in cortical neurons (CTX) than in cerebellar neurons (CBL). The basal level was low in the cerebellar neurons.
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Supplementary Figure 7
Inclusion bodies of p53 protect cortical neurons against mutant htt toxicity
(A) The panels show a representative neuron carrying p53 inclusion bodies (arrow) that keeps viability in trypan blue staining (TB). (B) The panel shows a quantitative analysis of the cell death percentages. The percentage of cell death was lower in inclusion positive cells than in cortical neurons (p < 0.01, Student’s t-test). In each case, nearly 100 neurons were analyzed.
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Supplementary Figure 8
A hypothetical scheme of molecular mechanisms classifying vulnerable and resistant neurons in the HD pathology
In this scheme, htt indicates the species in the pathogenic domains outside of inclusion bodies.
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Table 1
Table 1 shows selected genes whose expression was changed more than 3 folds. The values in F-K columns show the mean ratios of mRNA levels of the genes (column E) in each type of neurons (CTX: cerebral cortical neurons, STR: striatal neurons, CBL: vcerebellar neurons) under mutant polyQ protein expression and normal polyQ protein expression by adenovirus vectors. The blue background marks genes whose expression was decreased more than 3 folds by a mutant polyQ protein in comparison to the value by the normal polyQ protein expression. The red background indicates genes whose expression was upregulated more than 3 folds by a mutant polyQ protein. The F-H columns show the change under htt expression and the I-K columns show the change under Atx-1 expression. The yellow background marks genes whose signals were bellow 500 both in normal and mutant polyQ expression. The yellow marked genes are excluded from Figure 1B.
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Table 2
Table 2 shows raw data of mean signal values obtained from gene chip spots, in addition to the information listed in Table 1. The yellow background indicates the low signal bellow 500.
