Supplementary Figure 1. Independent Validation of Cwo As a Clock Component. Typical Locomotor

Supplementary Figure 1. Independent validation of cwo as a clock component. Typical locomotor activitiesof flies of over-expression of a dominant negative type of CWO protein. The names of strains and types of rhythmicity are described at each actogram.

Supplementary Figure 2. CWO expression in cwo RNAi and cwo deficient fly.Western blot analysis with antisera raised against CWO protein. The clear CWO signal was obtained at ~75kD in heads extracts in wild-type at ZT21 but obscure in cwo RNAi flies (A,C) and homozygous f05073 flies (B,D). C,D,The intensities of the bands were quantitated at the highest concentration by densitometry, and normalized so that the intensity of wild-type is 1.0.

Supplementary Figure 3. Circadian expression of Hes5 mRNA in mouse suprachiasmatic nucleus. Temporal expression profiles of Hes5 mRNA in mouse suprachiasmatic nucleus under LD and DD. Relative mRNA levels of each gene were measured with DNA chip (GeneChip) and quantitative PCR assay (Q-PCR), respectively. Tbp was used as an internal control. Error bars represent SEM (n=2).

Supplementary Figure 4. Circadian expression of cwo mRNA abolished in arrhythmic mutant flies. Temporal expression profiles of per, tim and cwo mRNA in per01 (red rectangle) and ClkJrk (black circle) mutant flies under LD and DD. Relative mRNA levels of each gene were measured with quantitative PCR assay. GAPDH2 was used as an internal control. Error bars represent SEM (n=2).

Supplementary Figure 5. pUAS-cwo-IR construct inhibited the translation of cwo. A, Western blot analysis with/without cwo knockdown construct (pUAS-cwo-IR) in S2 cells. Flag-CWO protein was reducedby thetransfection of pUAS-cwo-IR plasmid to S2 cells. B, Quantitative representation of the result shown in (A). The intensities of the bands were measured at the highest concentration by densitometry, and the relative intensities (Flag-CWO/ Flag-GFP ratio) in each sample were calculated. C, Expression of endogenous cwo mRNA inpUAS-cwo-IR transfected S2 cell. Relative mRNA levels of cwo in S2 cells transfected with the indicated plasmidsand pWAGAL4 were measured using a quantitative PCR assay. GAPDH2 was used as an internal control. Data were normalized so that the average copy number (n=2) of pUAST is 1.0. Error bars represent SEM (n=3). D, Expression of exogenous Flag-cwo mRNA in pUAS-cwo-IR transfected S2 cell. Relative mRNA levels of Flag-cwo mRNA in S2 cells transfected with the indicated plasmids were measured using a quantitative PCR assay. Exogenous Flag-GFP was used as an internal control. Data were normalized so that the average copy number (n=2) of pUAST is 1.0. Error bars represent SEM (n=3).