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Appendix S1

Figure S1: Microarray analysis of Clic4 expression and response to ethanol in PFC of C57BL6/J and DBA/2J mice. Robust multichip analysis (RMA) expression values for Clic4 probeset ID 94254_at are presented from data published in Kerns et al. (2005).

Figure S2: Representative time-course of negative geotaxis in the continuous presence of vapor in flies. eRING assays were performed in Control w1118 (○), ClicG0472/+ ( ) and ClicEY04209/+ (■) flies. Data from flies exposed to water vapor are shown at 0 (zero) min of ethanol exposure. Data from flies exposed to ethanol are shown at 1–11 min of ethanol exposure.

Figure S3: Ethanol sensitivity in additional studies with Drosophila Clic transposon mutants. Ethanol sensitivity (T50 values) from eRING studies in hemizygous males (a, b) and homozygous females (c, d) were reared at 20°C to bypass the lethality associated with strong loss of function in Clic and then tested at 25°C using standard conditions. In all panels the Clic mutants have higher T50 values than Control w1118 flies.

Figure S4: Rapid tolerance to ethanol is not affected in Drosophila Clic mutants. Rapid ethanol tolerance (calculated as [(T50EE/T50E − 1) × 100%] where T50E and T50EE are determined, respectively, during a first and second exposure to ethanol separated by 4 h of recovery without ethanol) in ClicG0472/+ (a) and ClicEY04209/+ (b) was not significantly altered compared to Control w1118 flies.

Figure S5: Locomotor (climbing) behavior in the absence of ethanol in Drosophila Clic mutants. (a, c) Climbing speed was not significantly altered in ClicG04722/+ (a) or ClicEY04209/+ (c) compared to Control w1118 flies. (b, d) Climbing latency (time to initiate climbing) was significantly shorter in ClicG04722/+ mutants compared to Control w1118 flies, but was not significantly altered in ClicEY04209/+ flies.

Figure S6: Initial sensitivity to ethanol and locomotor behavior in the absence of ethanol C. elegans N2 controls and Clic mutants. (a) Data from Fig. 5 replotted to show increased relative locomotor speed in exc-4(rh133) compared to N2 controls during the first 5 min of ethanol exposure. Other mutant genotypes are not shown forclarity. (b) Locomotor speed in the absence of ethanol in N2 control, exc-4(rh133), exl-1(ok857) and exc-4(rh133);exl-1(ok857). Data are from four independent experiments with 10 animals each per genotype.

Figure S7: Expression of Clic4 correlates with expression of RNA processing genes in mouse PFC. Microarray expression analysis of PFC from saline-treated BXD mouse strains (Wolen and Miles, unpublished) was used to identify genes having highly significant (FDR < 0.1%) Pearson correlations with Clic4 expression. Ingenuity Pathway Analysis (IPA) identified a highly significant overrepresentation of genes functioning in RNA processing. The network diagram displays the gene–gene interactions for this group of genes as determined by literature association analysis within IPA. Red and green coloration of gene symbol hubs reflect positive or negative correlations with Clic4 expression, respectively.

Table S1: Gene probesets having significant correlations with Clic4 expression in PFC of saline treated BXD strains. Data analysis is described in Methods. This gene list was used for functional over-representation within Ingenuity Pathway Analysis (Fig. S7).

Table S2: ToppGene over-representation analysis of genes with correlated basal expression with Clic4 across the BXD panel of mice in PFC. Genes were selected and analyzed for functional group over-representation as described in Methods.

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