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Fig. S1. U0126 treatment sensitizes zebrafish embryos for copper toxicity. (A) Low doses of copper chloride induced rapid death (y-axis, % viability) in zebrafish embryos already treated with U0126 for 24 hours, compared with DMSO- and CI-1040-treated embryos. (B) Zinc chloride had no effect on viability of U0126-treated embryos. (C) The lethal effects of exogenous copper on U0126-treated embryos were rescued by the addition of neocuproine (neo).
Fig. S2. Quality-control plots for barcode microarrays as listed in Table S1. (A) Boxplots of Z-scores for each of the microarrays showed that the quantile statistics were similar for all arrays; only the two JFD00692 experiments had a slightly different distribution. The compound-treated samples hybridized to these two arrays showed growth inhibition similar to other samples. (B-G) For each microarray, three quality-control plots are shown. Scatter plots of red (control) and green (treated) log2 intensities confirmed that most deletion mutants were not affected by compound treatment. Histograms for each microarray display intensity distribution in the two channels, which indicated even hybridization for both samples without any apparent dye bias. This analysis also revealed that a minor fraction of tags yielded low or no signal in both channels, owing to either mutated barcode sequences and/or stochastic variation in the composition of the strain pool.
Fig. S3. Heatmap of Z-scores for the specific genes within each GO category represented in Fig. 2B. Yeast mutants are sensitive (red) or resistant (green) to indicated compounds. Two different concentrations of each compound were screened. Annotation of genes with GO biological processes are taken from the Saccharomyces Genome Database (http://www.yeastgenome.org, release 02/07/2009).
Fig. S4. Neocuproine, CI-1040 and SEW01049 (40 mM solution) complex with copper as indicated by color change upon addition to 40 mM copper chloride solution. Droplets of the mixed solutions are presented on a microscope slide.
Fig. S5. Alignment of yeast Aps1 and Aps3 with zebrafish and human homologs. Zebrafish and humans have multiple homologs of yeast Aps1 and Aps3, including Ap1s1 and Ap3s2, respectively, in zebrafish, and AP1S1 and AP3S2, respectively, in humans (http://www.ensembl. org/Danio_rerio). (A) Alignment of budding yeast Aps1 with zebrafish Ap1s1 and human AP1S1 revealed 51% and 50% identity, respectively. (B) Alignment of budding yeast Aps3 with zebrafish Ap3s2 and human AP3S2 revealed 24% identity. Zebrafish Ap1s1 and human AP1S1 shared 91% identity, and zebrafish Ap3s2 and human AP3S2 shared 89% identity. Shading indicates homology for each residue: 100% similarity (black shading, white letters), 80% similarity (dark-grey shading, white letters) and 60% similarity (light-grey shading, black letters).
Fig. S6. Confirmation of altered RNA product by splice-site morpholino oligonucleotides. (A) Single-cell zebrafish embryos were injected with a splice-site mopholino ap3s2-MO1 targeting the splice junction of exon2 of ap3s2. Two days pf, embryonic RNA was extracted and subjected to PCR analysis. Purification and sequencing of the larger PCR product indicated the product had an inclusion of intron 2 (arrow). (B) PCR analysis of a second splice-site mopholino ap3s2-MO2 targeting the splice junction of exon 3 and intron 3 shows the presence of a smaller PCR product (arrow), consist with effective MO targeting. C. PCR analysis of morpholino ap1s1-MO2 targeting the acceptor splice site of intron 2 reveals the presence of a smaller transcript (Montpetit et al., 2008).
Fig. S7. Neocuproine does not inhibit phopho-ERK, but U0126 is an effective MEK inhibitor in human glioma cell lines. (A) U0126 treatment (10 µM and 200 µM) effectively inhibits phospho (p)-ERK signaling, whereas a control U0124 does not inhibit p-ERK. PD98059 treatment (20 µM and 200 µM) also inhibits p-ERK but less effectively than U0126. (B) Neocuproine treatment (10 µM and 100 µM) does not inhibit p-ERK signaling in human glioma cells.
