Hamamichi et al. 10.1073/pnas.0711018105.

Supporting Information

Files in this Data Supplement:

SI Table 3
SI Figure 3
SI Table 4
SI Figure 4
SI Figure 5
SI Table 5
SI Figure 6
SI Figure 7
SI Methods




SI Table 3



SI Figure 3

Fig. 3. An interconnectivity map summarizing the relationships between 17% (125/757) of the genes knocked down via RNAi that were nonlethal and classified in more than one bioinformatic category or cellular pathway.





SI Table 4



SI Figure 4

Fig. 4. RNAi knockdown of the top 20 gene targets did not enhance misfolding of polyglutamine aggregates in worms expressing Q82::GFP + TOR-2 in body wall muscle cells. (A) Isogenic worm strain expressing Q82::GFP + TOR-2 in C. elegans. (B) When worms expressing Q82::GFP + TOR-2 are exposed to tor-2 RNAi, the Q82::GFP aggregation returns. (C and D) When worms expressing Q82::GFP + TOR-2 are exposed to C35D10.2 or vps-41 RNAi, Q82::GFP aggregation is not enhanced. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded polyglutamine protein, as previously reported (1), and RNAi knockdown reverses this effect (B).

1. Caldwell GA, et al. (2003) Suppression of polyglutamine-induced protein aggregation in Caenorhabditis elegans by torsin proteins. Hum Mol Genet 12:307-319.





SI Figure 5

Fig. 5. Expression of α-syn in worm DA neurons results in age- and dose-dependent neurodegeneration. (A) Semiquantitative RT-PCR demonstrating that two separate isogenic worms strains expressing α-syn (low and high) have differing levels of mRNA when compared to the cdk-5 control. (B) Integrated transgenic line containing both Pdat-1::α-syn (high) and Pdat-1::GFP shows DA neurodegeneration over time, as animals age.





SI Table 5



SI Figure 6

Fig. 6. Analysis of transgene expression in worm strains. Semiquantitative RT-PCR was performed by using primers to amplify cdk-5 (control), α-syn (specific to the α-syn-expressing transgenic line), and primers specific to the clones analyzed. For all primers, N2 WT animals were used as both a positive (cdk-5) and negative (α-syn and transgene expression) control. Worms expressing α-syn without candidate PD transgenes were also analyzed where cdk-5 and α-syn primers were positive controls and primers corresponding to the transgenes were negative controls. The candidate PD transgenes were amplified by using gene-specific primers; all three separate transgenic lines for each clone were analyzed; α-syn primers were used as a negative control.





SI Figure 7

Fig. 7. Quantitative analysis of the hit rate of genes at both the primary and secondary level of RNAi screening compared with starting genes based on specific associations. (A) Candidates separated according to category (mechanisms: UPS, UPR, ERAD, or autophagy; worm bioinformatics: C. elegans microarray or interactome data; α-syn proteomic/genetics: proteomic or yeast genetic analyses). The highest hit rate came from genes that were coexpressed with a known PD gene and are associated with a cellular mechanism implicated in PD. (B) Candidates derived from microarray coexpression data for both DJ-1 and PINK1 are highly enriched at both the primary and secondary levels of RNAi screening for α-syn modifiers.





SI Methods