Supporting Information
Isojima et al. 10.1073/pnas.0908733106
SI Text
Detailed Procedures of the Screenings of Chemical Compounds That
Affect the Period Length Both in Mouse and Human Clock Cells. As

described in the main text, we examined 1,260 pharmacologically
active chemical compounds from the LOPAC1280 library (SigmaAldrich) (1). We first screened this library with relatively highconcentrations (25 ␮M for NIH 3T3-mPer2-Luc and 50 ␮M for
U2OS-hPer2-Luc) to reduce false negatives. We found 253
compounds for NIH 3T3-mPer2-Luc cells and 294 compounds
for U2OS-hPer2-Luc cells that either altered the period length
by more than 3 s.d. from the negative control or induced
apparent arrhythmicity. The intersection of these data yielded
166 compounds: Six and 40 compounds that shortened andlengthened, respectively, the period length of both cell types, and120 compounds that induced apparent arrhythmicity (including
compounds that were potentially cytotoxic or could affect the
LUC readout) in both cell types (Table S1).
To confirm the efficacy of the 166 compounds selected above,and to exclude potentially cytotoxic or LUC-readout altering
compounds, we conducted dose-dependency assays using thetwo clock cell lines. Three concentrations of each compound
were tested for period-altering effects of approximately one
order of magnitude from the concentration used in the primary
assay (25, 8.3, and 2.8 ␮M for NIH 3T3-mPer2-Luc; and 50, 16.7,
and 5.6 ␮M for U2OS-hPer2-Luc). If the lowest concentration of
a compound induced an apparent arrhythmicity in the cells, two
additional decreases in concentration were tested (0.93 and 0.31
␮M for NIH 3T3-mPer2-Luc; 1.9 and 0.62 ␮M for U2OS-hPer2Luc). This experiment identified nine compounds that significantly shortened (⬍-3 s.d.) and 30 that significantly lengthened(⬎⫹3 s.d.) the period length of NIH 3T3-mPer2-Luc cells, andseven and 90 compounds that shortened and lengthened, respectively, the period length of U2OS-hPer2-Luc cells (Table
S2). After integrating these data across the cell types, we found
four and 24 compounds that respectively shortened and lengthened the period of both cell lines. We designated these 28
compounds as ‘‘effective’’ compounds, and we believe they will
be a useful chemical resource for circadian biology (Table S3 andTable S4). We further examined these compounds to find thosewith the ‘‘potent’’ effects (⬎⫹10 s.d. or ⬍-10 s.d., ⬇2.0–3.0 h)
as described in the main text.
Chemical Landscape of Period Determination in Mammalian Circadian
Clocks. As described in the main text, a chemical-biologicalapproach was recently proposed to help elucidate the basic
processes underlying circadian clocks (2). Working on a relatively small scale, previous studies revealed that several protein
kinase inhibitors [such as IC261 (3, 4), CKI-7 (5), lithium
chloride (6, 7), SP600125 (4, 8), and SB203580 (9)], adenylate
cyclase inhibitors [such as THFA (9-tetrahydro-2-furyl-denine),2⬘5⬘-dideoxyadonosine, and 9-cyclopentyladenine (4)], and proteasome inhibitors [such as MG132 and lactacystin (3)], canlengthen the period of mammalian circadian clocks by 2% to⬇40%. Moreover, high-throughput screening of a large chemicalcompound library (1) supported using a chemical-biologicalapproach to probe the fundamental processes of the mammalian
circadian clock.
This study presents a chemical-biological approach to controlling, analyzing, and exploring the period-determination processes in the mammalian circadian clock. Using this approach, we
identified 28 chemical compounds that significantly (⬎3 s.d.)altered the period length of both human and mouse clock cell
Isojima et al. www.pnas.org/cgi/content/short/0908733106

lines (Table S3 and Table S4). Among them, 10 ‘‘potent’’compounds markedly (⬎10 s.d.) lengthened the period (Fig. S1
A and D), and lengthened it in both peripheral clock cells(MEFs) and central clock tissues (SCN) (Fig. S1 B, C, and E).
These results suggest that our protocol, which featured a highthroughput chemical screening strategy and multiple cell lines ofdifferent origin [NIH 3T3 and U2OS cells were derived from
NIH/Swiss mouse embryo cultures (10) and from human osteocarcinoma cells (11), respectively], and which applied a stringent
threshold for ‘‘potency,’’ enabled the identification of exceptionally potent chemical probes that target fundamental processes inmammalian circadian clocks. By probing this ‘‘chemical landscape,’’ we uncovered ‘‘potent,’’ ‘‘effective,’’ and even ‘‘ineffective’’ compounds, which will serve as a useful guide for drug
researchers seeking cures for rhythm disorders.
So far, this chemical landscape features at least two salient
landmarks: One is the period-determining process targeted by
17-OHP and the other is the period-determining process targeted by CKI␧/␦ activity. Since progesterone and progestins,
synthetic progesterone derivatives such as 17-hydroxyprogesterone caproate and medroxyprogesterone, are widely available asclinical drugs (12), the 17-OHP ‘‘landmark’’ serves as a good
vantage point for putative therapeutics (Fig. S1) to mitigate
rhythm disorders such as advanced sleep phase syndrome(ASPS) (i.e., via an agonist like 17-OHP). While guiding drug
discovery, the landmarks on this revealing chemical landscapewill also be useful for further exploration of the fundamental
properties of mammalian circadian clocks.
Validity of Relatively Higher-Dose Applications of Potent Effective
Compounds in Vitro and in Cellulo. In this chemical-biological study,

we discovered a highly flexible period-lengthening response in
the mammalian clock: A human clock cell’s circadian period
could be stretched into a circabidian period (Fig. 2 A) throughchemical manipulation, probably of the CKI activity, by
SP600125 and TG003. The half-maximum concentrations ofthese compounds for their period-lengthening effect in cellulo
were ⬇22 and 63 ␮M, respectively. In contrast, the IC50s of
SP600125 and TG003 for the CKI␧ kinase activity with 20 ␮M
ATP in vitro were ⬇0.22 and 0.55 ␮M, respectively (Fig. 1E). If
these compounds function as competitive inhibitors for ATP,
this apparent discrepancy can be largely explained by the difference in ATP concentration, which is ⬇1 mM inside cells(13–15). This competitive-inhibition hypothesis is experimentally supported by our observations that higher amounts ofinhibitors were required in assays with a high concentration of
ATP (100 ␮M ATP; Fig. 1D) than with a lower concentration (20␮M ATP; Fig. 1E). Given that the Michaelis constant for ATP
with CKI␧ is ⬇20 ␮M, the ratio of the IC50 with 1 mM ATP (incellulo) to that with 20 ␮M ATP (in vitro kinase assay) is 25.5,
and hence the IC50s of SP600125 and TG003 with 1 mM ATP
should be ⬇5.6 and 14.5 ␮M, respectively. Although there is stilla 4-fold difference between the half-maximum concentrations incellulo and the estimated IC50s with 1 mM ATP, these values arewithin the same order of magnitude. The difference might beowing to the dynamics of these compounds in cellulo, such as theefficiency of their penetration into cells, nonspecific buffering byother proteins within cells, and excretion from cells. Anotherpossibility is that these potent compounds act on other targets inaddition to CKI␧ and CKI␦, such as their ‘‘primary’’ targets,
which might be strongly involved in the period-determining
processes of mammalian circadian clocks. However, the latter1 of 12possibility was not supported by the results of our siRNA
experiments, in which we knocked down kinases including the‘‘primary’’ targets of the potent compounds (Fig. S2).
Temperature-Insensitivity of CKI Phosphorylation Depended on the
Substrate Recognition. CKIs recognize the motif ‘‘Ser/Thr-x-x-

Ser/Thr,’’ and phosphorylate the Ser/Thr residue at the Cterminal end. Negative charge at the N-terminal end of the motif
was thought to be the ‘‘priming signal’’ of the CKI-dependent
phosphorylation, because phosphorylation of the Ser/Thr residue at the N-terminal end or replacement of the residue to theacidic amino acid (Glu or Asp) remarkably improved the efficiency of the CKI-dependent phosphorylation (16). As describedin the main text, phosphorylation of ␤TrCP peptide or FASPSpeptide by ⌬CKI␧(wt) were relatively temperature-insensitive(␤TrCP; Q10 ⫽ 1.0, FASPS; Q10 ⫽ 1.2), although that of CK1tide
was more temperature-sensitive (CK1tide; Q10 ⫽ 1.4) (Fig. 3). It
should be noted that the CK1tide was ‘‘primed,’’ but the former
two substrates were not. Moreover, the tau mutation (R178C) of
CKI␧, which are thought to affect substrate recognition of CKI␧,
resulted to become temperature-sensitive in the phosphoryaltionof the FASPS-peptide (Q10 ⫽ 1.6). These results implied that
substrate recognition by CKI␧/␦ was a crucial process for defining temperature-dependency of CKI␧/␦-dependent phosphorylation. We also noted that priming phosphorylation at a Ser-662
residue of hPER2, associating with FASPS, was regulating
protein stability and nuclear translocation or transcriptional
repressor activity, although a kinase responsible for the priming
phosphorylation have not been identified (17).
SI Materials and Methods
Establishment of a High-Throughput Screening System Using a Cooled
CCD-Camera (CCD-Tron System). To perform high-throughput

screening of a chemical compound library, we developed ascreening system that uses a high-performance CCD camera,
based on a monitoring system using photomultiplier tubes(PMT) described in a previous report (PMT-Tron system) (18).
The round turntable (Mashinax or NEXSYS Corp. ) enables theobservation of up to 12 culture plates at once, and thus 4,608
samples can be screened simultaneously if 384-well culture plates
are used. The computer-controlled turntable rotates and sequentially sets each of the plates under the CCD camera every
5 min. The bioluminescence from the culture plate is imaged by
the high-performance CCD camera (VersArray XP or PIXIS;Roper Scientific). The intensity of each well is calculated fromthe obtained image by a custom-made computer program
Establishment of Stable Transfectants of NIH 3T3 Cells Expressing themPer2-Luc Reporter. The mouse Per2 promoter-driven reporter

plasmid (mPer2-Luc) (19) and the pIREShyg3 plasmid (BDBiosciences) were cotransfected into NIH 3T3 cells with FuGene6 transfection reagent (Roche). NIH 3T3 clones expressing
mPer2-Luc (NIH 3T3-mPer2-Luc) were selected using 200
␮g/mL hygromycin B (Invitrogen).
Establishment of Stable Transfectants of U2OS Cells Expressing thehPer2-Luc Reporter. A 334-bp fragment of the human Per2 genepromoter, 502- to 169-bp upstream from exon 1, was amplified
by PCR with PrimeSTAR DNA polymerase (TaKaRa) and thefollowing primers:
F: 5⬘-GGGGTACCGAGGGCGTAGTGAATGGAAG-3⬘
R: 5⬘-CCCAAGCTTAGCTGCACGTATCCCCTCAG-3⬘
This promoter fragment was subcloned into the reporter
vector, pGL4.14 (Promega) with the restriction enzymes, KpnIand HindIII (TaKaRa). This hPer2 promoter-driven reporter
plasmid (hPer2-Luc) was transfected into U2OS cells (purchasedfrom American Type of Culture Collections; ATCC), and stable
Isojima et al. www.pnas.org/cgi/content/short/0908733106

transfectants (U2OS-hPer2-Luc) were selected as described for
the NIH 3T3 cells.
Preparation of Embryonic Fibroblasts from mPer2Luc Mice. The
mPer2Luc mice (20) were carefully kept and handled according to
the RIKEN Regulations for Animal Experiments. Embryos ofmPer2Luc mice at E13 ⬇15 were excised and washed several times
with Hanks’ balanced salt solution. After the placenta andinternal organs were completely removed, the embryos were cut
into small pieces, and the cells were dissociated by incubation in
0.05% trypsin solution with 0.53 mM EDTA for 30 min at 37 °C.
The dissociated cells (mouse embryonic fibroblasts frommPer2Luc mice; mPer2Luc MEFs) were suspended and cultured in
the growth medium (DMEM supplemented with 10% FBS).
Preparation of SCN Slices from mPer2Luc Mice. The brains were

removed from quickly decapitated, young (older than 4 weeks ofage) mPer2Luc mice, then 300-␮m-thick coronal sections containing the SCN were made using a vibratome type linearslicer(PRO7; Dosaka EM Corp) in ice cold Hanks’ balanced salt
solution (Invitrogen). The slices were then placed on a culture
membrane (MilliCell-CM; Millipore) and set on a dish with 1.2
mL culture medium [DMEM supplemented with 1.2 g/L
NaHCO3 (Nacalai Tesque), 15 mM HEPES (Dojindo), 20 mg/L
kanamycin (Invitrogen), 5 ␮g/mL insulin (Sigma), 20 nM putrescine (Sigma), 100 ␮g/mL apo-transferrin (Sigma), 20 nM
progesterone (Sigma), 30 nM sodium selenite (Sigma), onefiftieth part B-27 supplement (Invitrogen), and 100 ␮M luciferin]
containing the chemical compounds at the concentrations indicated in Fig. S1C.
Analysis of the Effects of Compounds on Period Length in Cultured
Cells and Slices. NIH 3T3-mPer2-Luc cells, U2OS-hPer2-Luc cells,

and mPe2Luc MEFs were cultured on 24-well (PerkinElmer),
96-well, or 384-well (Falcon) culture plates, and 300 ␮m-thickcoronal slices containing the SCN from mPe2Luc mice on aculture membrane (MilliCell-CM; Millipore) in medium supplemented with 200 ␮M luciferin (Promega) and a chemicalcompound with (cells) or without (slices) stimulation with 10 nM
forskolin (Nacalai Tesque). The culture plates were placed in ahigh-sensitivity bioluminescence detection system (LM-2400;
Hamamatsu Photonics, for slices; PMT-Tron for MEFs; or
CCD-Tron, for others), and the bioluminescence of each well
was measured at 30 °C (cells) or 37 °C (slices) (Fig. S1 D and E).
The time-course bioluminescence data were analyzed as reported previously (18).
Rhythmicity and Period Length Analysis of Real-Time Bioluminescence
Data. The rhythmicity and period length were determined fromthe bioluminescence data of NIH 3T3-mPer2-Luc cells, U2OShPer2-Luc cells, MEFs, and SCN slices, as previously reported(18). Although this method could not be used for samples witha period length longer than 36 h, we modified the analysis
method to examine the long periods of circadian clocks in thepresence of high concentrations of potent period-lengthening
compounds (TG003 and SP600125) (Fig. 2 A). The data obtained
36 h after the addition of forskolin and compound, instead of
21 h, were used for analysis to eliminate the first cycle, anddetrending was performed not to detect the periodicity longer
than 72 h, instead of 42 h in usual method. These modifications
enabled us to examine periodicities up to 56 h.
Gene Knockdown Studies Using siRNA in U2OS-hPer2-Luc Cells.

U2OS-hPer2-Luc cells were plated at 3.0 ⫻ 104 cells per well in
24-well multiwell plates, 24 h before the transfection. The
predesigned Silencer Select siRNAs (Ambion) were transfectedwith Lipofectamine 2000 reagent (Invitrogen) as described in the
manufacturer’s protocol. At least three siRNA clones for each
2 of 12gene were examined. The siRNA was used at 5 to 20 pmol/well,and the total amount of transfected siRNA was adjusted to 20
pmol/well using Silencer Select negative control #2 siRNA(Ambion).
A pair of culture plates was prepared for each transfection
condition. The medium on one plate was changed to therecording medium containing 10 nM forskolin and 100 ␮M
luciferin 24 h after the transfection, placed into the PMT-Tronsystem, and the period length was measured as described above.
The total RNA of the cells on the other plate was purified with
TRIzol reagent (Invitrogen), and the cDNA was reversetranscribed using SuperScript II or III (Invitrogen) with random
primers (Invitrogen), as described in the manufacturer’s protocol. The gene expression levels were quantified with the QuantiFast SYBR PCR kit (Qiagen) with a LightCycler 480 (Roche
Applied Science) or PRISM 7900 HT (PerkinElmer), according
to the manufacturers’ protocols.
The ID numbers of the predesigned Silencer Select siRNAs
used in these studies were as follows:
Cry2. s3537; CSNK1E: s57, s58, and s59; CSNK1D: s3627, s3628,
and s3629; MAPK8: s11152, s11153, and s11154; MAPK9: s11158,
s11159, and s11160; CLK1: s3162, s3163, s3164; CLK2: s3165,
s3166, and s3167; CLK4: s32986, s32987, and s32988; CDK2:
s204, s205, and s206; CDK5: s2825, s2826, and s2827; CDC2:
s463, s464, and s465: MAPK14: s3585, s3586, and s3587;
MAPK11: s11155, s11156, and s11157; MAPK12: s12467, s12468,
and s12469; CSNK2A1: s3636, s3637, and s3638; CSNK2A2:
s3639, s3640, and s3641; ADORA1: s1085, s1086, and s1087;
ADORA2A: s1088, s1089, and s1090; ADORA2B: s1091, s1092,
and s1093. In addition, the following custom designed Silencer
Select siRNAs were used:
CLK1: s229161 and s229162; CLK2: s229159 and s229160;
CLK4: s229157 and s221958.
Expression and Purification of mCKI␧/␦ Protein. Mouse CKI␧ ORFs,

corresponding to amino acids 2–416 (full-length CKI␧) and
2–319 (⌬CKI␧), and CKI␦ ORFs, corresponding to amino acids
2–415 (full-length CKI␦) and 2–317 (⌬CKI␦), were cloned into
the pGEX-6P-1 vector (GE Healthcare) and then introduced
into Escherichia coli Rosetta2 (Novagen). A CKI␧ ORF in whicheight serine residues identified as autophosphorylation aminoacids (21) in the C-terminal region were replaced with glutamate, referred to as CKI␧ (D8), was also cloned and introduced
into E. coli. Cells were grown at 25 °C until they reached an
OD600 absorbance of about 0.5. After the addition of isopropyl
␤-D-1-thiogalactopyranoside (IPTG) to a final concentration of
0.1 mM, the cells were further grown at 25 °C for 24 h. The cells
were collected and resuspended in extraction buffer [50 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 10%
glycerol, and complete protease inhibitor mixture (Roche)] andhomogenized by sonication. The homogenate was spun at
38,000⫻ g, and the supernatant was applied to a Glutathione
Sepharose 4B column (GE Healthcare). The column was washedwith five column volumes of the buffer. PreScission Protease(GE Healthcare) was then applied to the column to remove the
GST-tag, according to the manufacturer’s protocol. The proteins
were eluted with two column volumes of elution buffer (50 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, and
10% glycerol), and the eluent was diluted and applied to a
HiTrap SP HP column (GE Healthcare). The proteins were theneluted with 600 mM NaCl. The protein concentration wasdetermined by the Bradford method, using the Bio-Rad Laboratories protein assay kit with BSA (Bio-Rad Laboratories) as astandard. The purified proteins used in this study are shown in
Fig. S6 B and C.
Isojima et al. www.pnas.org/cgi/content/short/0908733106

Measurement of CKI Enzymatic Activity. ⌬CKI␧ protein (0.5 ␮M)
was added to the reaction buffer (25 mM Tris-HCl, pH 7.5, 7.5
mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, and 200 nM synthetic
peptide substrate) with different concentrations of chemicals(25, 50, or 100 ␮M for the potent compounds; 100 ␮M for
IC261), preincubated at 30 °C for 5 min, and then the reaction
was started by adding a one-fourth volume of 400 ␮M ATP.
Aliquots (1 ␮L) of the reaction mixture were withdrawn in
triplicate at 0 and 1 h of the reaction and mixed with 30 ␮L IMAP
binding buffer in 384-well glass-bottomed plates (Olympus).
After incubating for at least 1 h at ambient temperature, thefluorescent polarization was measured using a single-molecule
fluorescence detection (SMFD) system (MF20; Olympus).
The measurement of CKI kinase activity using a P81 phosphocellulose paper assay was performed as follows. Peptides
consisting of 34 aa (RKKKPHSGSSGYGSLGSNGSHEHLMSQTSSSDSN), referred to as ␤TrCP-peptide (22),and 29 aa (RKKKTEVSAHLSSLTLPGKAESVVSLTSQ), referred to as FASPS-peptide, were synthesized and purchasedfrom Bio-Synthesis Inc. CK1tide (KRRRAL[pS]VASLPGL;
Upstate), and ␣-casein from bovine milk (Sigma) were used as
the substrate.
⌬CKI␧/␦ proteins (0.2 ␮M for ␤TrCP-peptide and FASPSpeptide, 0.02 ␮M for ␣-casein, 0.01 ␮M for CK1tide) in thereaction buffer (25 mM Tris, pH 7.5, 7.5 mM MgCl2, 1 mM DTT,
0.1 mg/mL BSA, and 200 ␮M peptide substrate or 40 ␮M casein)
were preincubated for 5 min. The reaction was started by adding
a one-fourth volume of 2 mM (Fig. S6D), 400 ␮M (Fig. 1D), or80 ␮M (Fig. 1E) ATP with 0.5–1 ␮Ci [␥-32P] ATP per reaction.
In assays for autophosphorylated CKI␧/␦ (0.4 ␮M for ␤TrCPpeptide, 0.04 ␮M for ␣-casein, and CK1tide), the enzyme waspreincubated with buffer (25 mM Tris, pH 7.5, 7.5 mM MgCl2,
1 mM DTT, 0.1 mg/mL BSA, 500 ␮M ATP) at 30 °C for 1 h, andthe reaction was started by adding substrate. The preincubationand incubation steps were performed at 25 ° or 35 °C. Twoaliquots (10 ␮L) of the reaction mixture were withdrawn at eachtime point and spotted onto P81 phosphocellulose paper (Whatman) in the case of peptide substrates, or mixed with SDS-PAGE
buffer for casein. The P81 phosphocellulose papers were washedfive times with 75 mM orthophosphate and once with 100%
EtOH, then placed in scintillation vials with scintillation mixture(AQUASOL-2; PerkinElmer). The incorporation of Pi wasquantified using a scintillation counter (LSC-6100; Aloka) or byautoradiography after SDS-PAGE (BAS-2500; Fuji Photo Film).
Analysis of the Degradation Rate of mPER2 Protein in 293T, NIH 3T3,
or mPer2Luc MEF Cells. The intensity of the LUC::mPER2 from293T cells, mPER2::LUC from NIH 3T3 cells, or mPer2Luc
MEFs was normalized so that the average luminescence detected
at t ⫽ 0 was defined as ’100⬘. To calculate the half-life of
LUC-fused protein, the normalized results were fitted with theequation,y ⫽ ae⫺kt ⫹ 共100 ⫺ a兲y, normalized bioluminescence intensity; t, time after addition of
cycloheximide (CHX); a, k, variables. e, Napier’s constant.
And the half-life t1/2 was calculated as follows:
t1/2 ⫽ ⫺ln冉冊

共a ⫺ 50兲 1.
akAnalysis of the Temperature Sensitivity of mPER2 Degradation in themPer2Luc MEFs. The cultures were maintained at 27 °, 32 °, or 37 °C

for 4 days, and the bioluminescence from the cells was monitoredby the PMT-Tron system until the oscillations of the bioluminescence from mPER2::LUC triggered by the temperaturechange were diminished. The cells were then treated with 400
3 of 12␮g/mL CHX. The bioluminescence was recorded every 30 min
for 5 h by the PMT-Tron system. The time course of thebioluminescence in each well was normalized and analyzed as
described for the degradation rate of the LUC::mPER2 protein
in 293T cells.

cated times after adding 200 ␮g/mL CHX. The LUC activity wasmeasured using the Dual-Glo bioluminescence detection kit(Promega) following the manufacturer’s protocol. The degradation rate was calculated as described above.
Western Blot of mPER2 Protein Expressed in 293T Cells. 293T cells,

Construction of Expression Vectors for a Reporter Assay to Examine
Clock-Related Protein Stability. To investigate the stability ofclock-related proteins (PER2, PER1, and BMAL1) in cultured
293T cells, we constructed an expression plasmid bearing thefirefly Luciferase protein fused to the N-terminal end of clockrelated proteins (LUC::mPER2, LUC::mPER1, and
LUC::mBMAL1) under the CMV promoter (CMV-Luc::mPer2,
CMV-Luc::mPer1, and CMV-Luc::mBmal1). We also constructed an expression vector bearing the firefly luciferase
protein fused to the C-terminal end of the mouse Per2 protein(mPER2::LUC) using a custom expression vector pMU2 (18) for
studies in cultured NIH 3T3 cells (pMU2-mPer2::Luc). The
cDNAs of these clock-related proteins were kindly provided by
Dr. H. Tei (Mitsubisi Kagaku Institute for Life Sciences, Japan).
A control expression vector expressing the LUC protein under
the CMV promoter (CMV-Luc) was also constructed.
The coding sequence of mCKI␧(wt) was subcloned into thepMU2 expression vector as previously described [pMU2mCKI␧(wt)]. An expression vector for the mCKI␧(tau) mutant(in which Arg-178 was replaced with Cys) was constructed byinverse PCR using pMU2-mCKI␧(wt) as a template. Inverse
PCR was performed using the following PCR primers.
F: 5⬘-TGCTATGCCTCTATCAACACCCAC-3⬘
R: 5⬘-GGCAGTGCCGGTCAGGTTCTTG-3⬘ (the 5⬘ end
was phosphorylated).
Finally, self-ligation of the PCR product was performed. The
resultant vector was named pMU2-mCKI␧(tau).
An expression vector bearing only the catalytic domain of
mCKI␧ (⌬mCKI␧) was also constructed. The coding sequence ofthe regulatory domain of mCKI␧, corresponding to amino acids
320 to 416, was deleted from pMU2-mCKI␧(wt) by inverse PCR.
The resultant vector was named pMU2-⌬mCKI␧(wt).
The mCKI␧(wt), mCKI␧(tau), or ⌬mCKI␧(wt) gene was fusedin-frame with 1⫻ Flag Tag at the N terminus, and regulated by
the CMV promoter.

grown in DMEM supplemented with 10% FBS and antibiotics(25 U/mL penicillin, 25 mg/mL streptomycin; Invitrogen), wereplated at 4 ⫻ 105 cells per dish in 35-mm dishes. Twenty-fourhours later, the cells were transfected with FuGene6 (Roche),according to the manufacturer’s instructions. The cells weretransfected with 2 ␮g pcDNA3-Myc-mPer2 (a gift from Dr. E.
Nishida, Kyoto University) (23), 2 ␮g pMU2-mCKI␧(tau), or
empty plasmid pMU2, and 2 ␮g pMU2-Luc. After 24 h, the cells
transfected with the mCKI␧(tau) expression vector were harvested, combined, and then replated onto the same number of
35-mm dishes to normalize for variations in the transfection
efficiencies due to different plates. After 48 h, the cells weretreated with or without 100 ␮M TG003 or SP600125 in 0.2%
DMSO (Nacalai Tesque). The treated cells were harvested at thetimes indicated in Fig. S4, lysed with the extraction reagent [50mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, Halt Protease
Inhibitor Mixture, EDTA-free (Pierce)], and sonicated. The
protein concentration of the samples was measured by the
Bradford method using a Bio-Rad Laboratories protein assay kit(Bio-Rad Laboratories). Equal amounts of protein (0.25 mg)
were then mixed with NuPAGE LDS sample buffer (Invitrogen),and the samples were separated by electrophoresis and transferred to PVDF membranes using the NuPAGE electrophoresis
system (Invitrogen), as described in the manufacturer’s protocol.
Myc-mPER2 protein was detected with an anti-Myc monoclonal
antibody (clone A-14; Santa Cruz Biotechnology.) and HRPconjugated anti-rabbit IgG secondary antibody (GE Healthcare). An anti-tubulin␣ monoclonal antibody (clone DM1A;LAB VISION) and anti-mouse IgG secondary antibody (GEHealthcare) were used to detect tubulin␣ protein. The immunoreactivities were visualized and quantified using the ECL Plus
Western Blotting Detection system (GE Healthcare) and LAS1000 (Fujifilm), according to the manufacturers’ instructions.
Analysis of Temperature Sensitivity of mPER2 or LUC Degradation in
NIH 3T3 Cells. NIH 3T3 cells cultured on 100-mm plates werefected with expression vectors for LUC::mPER2, CKI␧(tau), andRenilla Luciferase were subcultured on 96- or 384-well culture
plates. The stability of the LUC::mPER2 protein was measured
after a 24-h incubation with a chemical compound, and thedegradation rate of LUC::mPER2 was determined at the indi-transfected with 30 ␮g reporter vector (pMU2-mPer2::Luc or
pMU2-Luc) and 6 ␮g pMU2-⌬CKI␧(wt) or empty pMU2 using
60 ␮L FuGene6. The transfected cells were dissociated by trypsin
and plated on 24-well culture plates 24 h after transfection, andthe degradation of mPER2::LUC or LUC was measured as
described above for mPer2Luc MEFs.

1. Hirota T, et al. (2008) A chemical biology approach reveals period shortening of the
mammalian circadian clock by specific inhibition of GSK-3beta. Proc Natl Acad Sci USA
105:20746 –20751.
2. Liu AC, Lewis WG, Kay SA (2007) Mammalian circadian signaling networks and therapeutic targets. Nat Chem Biol 3:630 – 639.
3. Eide EJ, et al. (2005) Control of mammalian circadian rhythm by CKIepsilon-regulated
proteasome-mediated PER2 degradation. Mol Cell Biol 25:2795–2807.
4. O’Neill JS, et al. (2008) cAMP-dependent signaling as a core component of the mammalian circadian pacemaker. Science 320:949 –953.
5. Vanselow K, et al. (2006) Differential effects of PER2 phosphorylation: Molecular basis forthe human familial advanced sleep phase syndrome (FASPS). Genes Dev 20:2660 –2672.
6. Abe M, Herzog ED, Block GD (2000) Lithium lengthens the circadian period of individual suprachiasmatic nucleus neurons. Neuroreport 11:3261–3264.
7. Iwahana E, et al. (2004) Effect of lithium on the circadian rhythms of locomotor activity
and glycogen synthase kinase-3 protein expression in the mouse suprachiasmatic
nuclei. Eur J Neurosci 19:2281–2287.
8. Chansard M, et al. (2007) c-Jun N-terminal kinase inhibitor SP600125 modulates theperiod of mammalian circadian rhythms. Neuroscience 145:812– 823.
9. Hayashi Y, et al. (2003) p38 mitogen-activated protein kinase regulates oscillation ofchick pineal circadian clock. J Biol Chem 278:25166 –25171.
10. Jainchill JL, Aaronson SA, Todaro GJ (1969) Murine sarcoma and leukemia viruses: assay
using clonal lines of contact-inhibited mouse cells. J Virol 4:549 –553.

11. Ponten J, Saksela E (1967) Two established in vitro cell lines from human mesenchymal
tumours. Int J Cancer 2:434 – 447.
12. Meis PJ (2005) 17 Hydroxyprogesterone for the prevention of preterm delivery. Obstet
Gynecol 105:1128 –1135.
13. Wang RH, Tao L, Trumbore MW, Berger SL (1997) Turnover of the acyl phosphates ofhuman and murine prothymosin alpha in vivo. J Biol Chem 272:26405–26412.
14. Sippel CJ, Dawson PA, Shen T, Perlmutter DH (1997) Reconstitution of bile acidtransport in a heterologous cell by cotransfection of transporters for bile acid uptake
and efflux. J Biol Chem 272:18290 –18297.
15. Kennedy HJ, et al. (1999) Glucose generates sub-plasma membrane ATP microdomains
in single islet beta-cells. Potential role for strategically located mitochondria. J Biol
Chem 274:13281–13291.
16. Flotow H, et al. (1990) Phosphate groups as substrate determinants for casein kinase I
action. J Biol Chem 265:14264 –14269.
17. Xu Y, et al. (2007) Modeling of a human circadian mutation yields insights into clock
regulation by PER2. Cell 128:59 –70.
18. Ukai H, et al. (2007) Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell Biol 9:1327–1334.
19. Sato TK, et al. (2006) Feedback repression is required for mammalian circadian clockfunction. Nat Genet 38:312–319.
20. Yoo SH, et al. (2004) PERIOD2::LUCIFERASE real-time reporting of circadian dynamics
reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci
USA 101:5339 –5346.

Analysis of mPER2 Protein Stability in 293T Cells. 293T cells trans-

Isojima et al. www.pnas.org/cgi/content/short/0908733106

4 of 12

21. Gietzen KF, Virshup DM (1999) Identification of inhibitory autophosphorylation sites
in casein kinase I epsilon. J Biol Chem 274:32063–32070.
22. Lowrey PL, et al. (2000) Positional syntenic cloning and functional characterization of
the mammalian circadian mutation tau. Science 288:483– 492.
23. Akashi M, Tsuchiya Y, Yoshino T, Nishida E (2002) Control of intracellular dynamics ofmammalian period proteins by casein kinase I epsilon (CKIepsilon) and CKIdelta in
cultured cells. Mol Cell Biol 22:1693–1703.
24. Meijer L, et al. (1997) Biochemical and cellular effects of roscovitine, a potent andselective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem
243:527–536.
25. Muraki M, et al. (2004) Manipulation of alternative splicing by a newly developed
inhibitor of Clks. J Biol Chem 279:24246 –24254.
26. Bain J, et al. (2007) The selectivity of protein kinase inhibitors: A further update.
Biochem J 408:297–315.
27. Kummer JL, Rao PK, Heidenreich KA (1997) Apoptosis induced by withdrawal of trophic
factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem 272:20490 –
20494.
28. Fong TA, et al. (1999) SU5416 is a potent and selective inhibitor of the vascularendothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis,tumor vascularization, and growth of multiple tumor types. Cancer Res 59:99 –106.
29. Zandomeni R, Weinmann R (1984) Inhibitory effect of 5,6-dichloro-1-beta-Dribofuranosylbenzimidazole on a protein kinase. J Biol Chem 259:14804 –14811.
30. Ghai G, et al. (1987) Pharmacological characterization of CGS 15943A: A novel nonxanthine adenosine antagonist. J Pharmacol Exp Ther 242:784 –790.
31. Kim YC, Ji XD, Jacobson KA (1996) Derivatives of the triazoloquinazoline adenosine
antagonist (CGS15943) are selective for the human A3 receptor subtype. J Med Chem
39:4142– 4148.
32. Kraichely DM, Sun J, Katzenellenbogen JA, Katzenellenbogen BS (2000) Conformational changes and coactivator recruitment by novel ligands for estrogen receptoralpha and estrogen receptor-beta: Correlations with biological character and distinct
differences among SRC coactivator family members. Endocrinology 141:3534 –3545.
33. Ashley RL, et al. (2006) Cloning and characterization of an ovine intracellular seventransmembrane receptor for progesterone that mediates calcium mobilization. Endocrinology 147:4151– 4159.
34. Baker ME, et al. (1984) Chick oviduct progesterone receptor binding of 15 beta,
17-dihydroxyprogesterone and its analogues. Steroids 43:153–158.
35. Nelson EM, Tewey KM, Liu LF (1984) Mechanism of antitumor drug action: Poisoningof mammalian DNA topoisomerase II on DNA by 4⬘-(9-acridinylamino)-methanesulfonm-anisidide. Proc Natl Acad Sci USA 81:1361–1365.
36. Eagling VA, Tjia JF, Back DJ (1998) Differential selectivity of cytochrome P450 inhibitors
against probe substrates in human and rat liver microsomes. Br J Clin Pharmacol
45:107–114.

Isojima et al. www.pnas.org/cgi/content/short/0908733106

37. Yoon KW, Covey DF, Rothman SM (1993) Multiple mechanisms of picrotoxin block of
GABA-induced currents in rat hippocampal neurons. J Physiol 464:423– 439.
38. Smith DG, et al. (2001) 3-Anilino-4-arylmaleimides: Potent and selective inhibitors ofglycogen synthase kinase-3 (GSK-3). Bioorg Med Chem Lett 11:635– 639.
39. Pilla M, et al. (1999) Selective inhibition of cocaine-seeking behaviour by a partial
dopamine D3 receptor agonist. Nature 400:371–375.
40. Johnson RA, et al. (1997) Isozyme-dependent sensitivity of adenylyl cyclases to P-sitemediated inhibition by adenine nucleosides and nucleoside 3⬘-polyphosphates. J Biol
Chem 272:8962– 8966.
41. Thompson RD, Secunda S, Daly JW, Olsson RA (1991) Activity of N6-substituted 2-chloroadenosines at A1 and A2 adenosine receptors. J Med Chem 34:3388 –3390.
42. Kemppainen JA, et al. (1999) Distinguishing androgen receptor agonists and antagonists: Distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone. Mol Endocrinol 13:440 – 454.
43. Jacobson KA, et al. (1995) Structure-activity relationships of 9-alkyladenine and ribosemodified adenosine derivatives at rat A3 adenosine receptors. J Med Chem 38:1720 –1735.
44. Meyers MJ, et al. (2001) Estrogen receptor-beta potency-selective ligands: Structureactivity relationship studies of diarylpropionitriles and their acetylene and polar
analogues. J Med Chem 44:4230 – 4251.
45. Nymark M, et al. (1973) Prolonged neuroleptic effect of alpha-flupenthixol decanoate
in oil. Acta Pharmacol Toxicol (Copenh) 33:363–376.
46. Faust R, et al. (2000) Mapping the melatonin receptor. 6. Melatonin agonists andantagonists derived from 6H-isoindolo[2,1-a]indoles, 5,6-dihydroindolo[2,1a]isoquinolines, and 6,7-dihydro-5H-benzo[c]azepino[2,1-a]indoles. J Med Chem
43:1050 –1061.
47. Garvin JL, Simon SA, Cragoe EJ, Jr, Mandel LJ (1985) Phenamil: An irreversible inhibitorof sodium channels in the toad urinary bladder. J Membr Biol 87:45–54.
48. Kennett GA, et al. (1996) In vitro and in vivo profile of SB 206553, a potent 5-HT2C/5HT2B receptor antagonist with anxiolytic-like properties. Br J Pharmacol 117:427– 434.
49. Lal B, et al. (1984) Trequinsin, a potent new antihypertensive vasodilator in the seriesof 2-(arylimino)-3-alkyl-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-py rim ido [6,1a]isoquinolin-4-ones. J Med Chem 27:1470 –1480.
50. Knauf U, Tschopp C, Gram H (2001) Negative regulation of protein translation by
mitogen-activated protein kinase-interacting kinases 1 and 2. Mol Cell Biol 21:5500 –5511.
51. Showalter VM, Compton DR, Martin BR, Abood ME (1996) Evaluation of binding ina transfected cell line expressing a peripheral cannabinoid receptor (CB2): Identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther
278:989 –999.

5 of 12

D

SB202190

SU5416SP600125

PPT

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

TG003

PD169316

DRB

CGS-15943

17-OHP

Bioluminescence

40
36
32
28
24

20

50

0
10

0
70

0
10

0
60

0

2

0
48

96

0

48

0 10 20 30 40 50

0 10 20 30 40 50

B

Luc

mPer2
Roscovitine

MEF

SU5416

SB202190

SP600125(A.U.)

0 10 20 30 40 50

Bioluminescence

NIH3T30 10 20 30 40 50

96 144

0
0

48

0

96

NIH3T3

U2OS
20

80

2
12

0
100

0
16

0
70

2

0
0

48

96

0

96 144

U2OS

60

2

48

PD169316

20

PPT

40

U2OS

60

SB202190

Concentration (µM)

48

96 144

0
0

48

96

0

48

96 14436

0 5 10 15 20 25

0 5 10 15 20 25

TG003

PD16931640

0

5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

CGS-15943

17-OHP

DRB

20

36

32

28

24
0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

18

60

4
12

0
60

2
12

0
70

2
48

SCN

SU5416SP600125

PPT

32

32

Period Length (h, real)

28

24

24
0 20 40 60 80 100 0 20 40 60 80 100

TG003

PD169316

DRB

0 5 10 15 20 25

0 20 40 60 80 100

CGS-15943

17-OHP

32

32

28

28

24
5 10 15 20 25

0

5 10 15 20 25

0 5 10 15 20 25

10
0

96 14448

0 5 10 15 20 25

96

NIH3T3

U2OS
24

60

2
16

0
60

4
14

10
60

2

2

0
48

96

0

0 5 10 15 20 25

48

96 14448

96

0

NIH3T3

U2OS

60

2
16

0
50

0
8

0
50

0

0

2.8 µM
8.3 µM
25.0 µM

96

96 144

U2OS

14

48

48

17-OHP60

0

96 144

10
0

24

2

48

U2OS

60

NIH3T3

Concentration (µM)

0

CGS-15943

PPT

24
0

48

30

0

Bioluminescence28

0 5 10 15 20 25

0

NIH3T3(A.U.)

SB202190

96

SP600125
Bioluminescence

Roscovitine

2

10
0

(A.U.)

Luc

mPer2

U2OS

50

Concentration (µM)

C

NIH3T3

U2OS

18

20

0 5 10 15 20 25

DRB

NIH3T3

Bioluminescence28

24SU5416

Period Length (h, real)32

Period Length (h, CT)

TG003
NIH3T3

U2OS

20

0

40
36
32
28
24
0 10 20 30 40 50

Period Length (h, CT)

Roscovitine
NIH3T3(A.U.)

Period Length (h, CT)

NIH3T3-mPer2-Luc / U2OS-hPer2-Luc
Roscovitine(A.U.)

A

0

48

96 1445.6 µM
16.7 µM50.0 µM

0
0

48

96

0

48

96 144

Time (hr)

E
TG003
MEF

SCN
80000

0

0
0

60

120

30000

0
0 48 96 144 192

060

SB202190
MEF

MEF

80000

4000

0

60

120

SCN
120000

7000

10000

0

30000

0
0 48 96 144 192

0

60

(cpm)

Bioluminescence

MEF

DRB
MEF

SCN
80000

0

60

20000

0
0 48 96 144 192

120

0

60

MEF
(cpm)

Bioluminescence

SP600125
MEF

80000

0
60

20000

0
0 48 96 144 192

120

0

60

MEF
(cpm)

Bioluminescence

PPT
4000

MEF

SCN

6.25 µM
12.5 µM
25.0 µM50.0 µM
100.0 µM

60

120

SCN

4000

30000

0

70000
0

0 48 96 144 192

120

17-OHP

250000

0

SCN
90000

6000

20000
0

0 48 96 144 192

120

CGS-15943
SCN

8000

SCN
110000

4000

10000

0

0 48 96 144 192

120

SU54164000

0 48 96 144 192

120

PD169316
SCN

(cpm)

Bioluminescence

SCN
110000

5000

(cpm)

Bioluminescence

Roscovitine
MEF
6000

0 48 96 144 192

15000
0

60

120

0 48 96 144 192

Time (hr)Fig. S1. Dose-response of period length in the NIH 3T3-mPer2-Luc and U2OS-hPer2-Luc cells (A), primary cultures of MEFs (B), slice cultures of SCN (C) frommPer2Luc mice, and raw bioluminescence data (D and E). The period length were represented both in real time (right axis in B and C) and in circadian time (i.e.,
the period length of control samples was adjusted to 24 h, left axis). Blue circles in (A) represented average of NIH 3T3 (n ⫽ 2) and red squares U2OS (n ⫽ 3). Eachline represented independent experiments. Each value in (B and C) represents the mean ⫾ SEM. At the concentrations without data points, the cells behaved
arrhythmically. (D) Time course of the reporter activity in NIH 3T3-mPer2-Luc cells (left panels) and U2OS-hPer2-Luc cells (right panels) after forskolin stimulationand the addition of compound. The gray lines represent the average of control samples (n ⫽ 768 for NIH 3T3, and n ⫽ 96 for U2OS) and colored lines represent
the averages of samples containing compounds at the concentrations indicated at the base of the figure (n ⫽ 2 for NIH 3T3, and n ⫽ 3 for U2OS). The compound
name is displayed at the top of each set of four panels. The upper and lower panels within each group indicate separate experiments. (E) Time course of thereporter activity in MEFs (left panels) and SCN slices (right panels) after stimulation with compound with (MEFs) or without (SCN) forskolin. The gray lines
represent the average of control samples (n ⫽ 5 for MEF, and n ⫽ 4 for SCN) and colored lines represent the average of samples containing compound at theconcentrations indicated at the bottom of the figure (n ⫽ 3 for MEF, and n ⫽ 4 for SCN). The compound represented in the graphs is written above each pairof panels.

Isojima et al. www.pnas.org/cgi/content/short/0908733106

6 of 12

Fig. S2. Effects of gene knockdowns on the period length in U2OS-hPer2-Luc cells. (A) Relative expression levels of CKI␧, CKI␦ and genes related to the 10 potent
chemical compounds that lengthened the period length (see Table S3), examined by the quantitative real-time PCR method. The graph indicates the average⫹ SEM of the gene expressions relative to GAPDH (n ⫽ 6). The expression of the FLT1 gene was undetectable. (B–G) Relationship between gene knockdown effects
and period length in U2OS-hPer2-Luc cells. Two sets of U2OS-hPer2-Luc cells were transfected with predesigned Silencer Select siRNA: One was cultured on the
PMT-Tron system for measurement of the period length, and the other was analyzed for gene expression levels 24 h after the transfection. The x-axis indicatesthe expression level of genes relative to the samples transfected with control siRNA. The y-axis indicates the period length, described in circadian time (CT), withthe control samples assigned as 24 h. Each symbol represents the mean ⫾ SEM. of independent experiments (n ⱖ 3). (B) Gene knockdowns of kinases inhibitedby SP600125. MAPK8, mitogen-activated protein kinase 8 [MAPK8, also c-Jun N-terminal kinase 1 (JNK1)]; MAPK9, MAPK9 (JNK2). (C) Gene knockdowns of kinases
inhibited by TG003. CLK1, CDC-like kinase 1 (CLK1); CLK2, CLK2; CLK4, CLK4. (D) Gene knockdowns of kinases inhibited by Roscovitine. CDK1, Cyclin-dependent
kinase 1 (CDK1); CDK2, CDK2; CDK4, CDK4. (E) Gene knockdowns of kinases inhibited by SB202190 and PD169316. MAPK14, MAPK14 [also p38 MAPK ␣ (p38␣)];
MAPK11, MAPK11 (p38␤); MAPK12, MAPK12 (p38␥). (F) Gene knockdowns of kinases inhibited by DRB. CSNK2A1, CK2␣; CSNK2A2, CK2␣⬘. (G) Gene knockdownsof adenosine receptors inhibited by CGS-15943. ADORA1, Adenosine A1 receptor; ADORA2A, Adenosine A2a receptor; ADORA2B, Adenosine A2b receptor

Isojima et al. www.pnas.org/cgi/content/short/0908733106

7 of 12

Fig. S3. Effect of the potent effective compounds on the stability of mPER2 proteins in cellulo. (A) mPER2 stabilization in 293T cells. Bioluminescence from 293T
cells cotransfected with a reporter (LUC::mPER2 or LUC) and CKI␧(tau) was measured, and the ratio of the bioluminescence of LUC::mPER2 to LUC was plotted.
Each value represents the mean ⫾ SEM. (n ⫽ 16 for 0 ␮M, n ⫽ 4 for chemically treated samples). All of the compounds except 17-OHP exhibited a significant
dose-dependent effect (P ⬍ 0.01; one-way ANOVA). (B) mPER2 degradation rate in 293T cells. The averages (n ⫽ 3; closed circles) of the normalizedbioluminescence of 293T cells cotransfected with the LUC::mPER2 reporter and CKI␧(tau) after treatment with CHX in the presence (green) or absence (gray) ofa chemical compound were plotted. The fitted curve is also indicated in each panel. The time course of all of the samples except 17-OHP was significantly different
from that of the control samples (P ⬍ 0.001; two-way ANOVA). (C) The potent inhibitors dominantly affected the CKI-dependent degradation of LUC::mPER2
but not the CKI-independent degradation. The average ⫾ SEM. (n ⫽ 3) of the normalized bioluminescent activities of 293T cells cotransfected with the
LUC::mPER2 reporter and CKI␧(tau) (right panel) or control vector (left panel) with 100 ␮M SP600125 (red squares), TG003 (blue diamonds), or solvent only (0.25%
DMSO; gray circles) after treatment with CHX was plotted. The fitted curve is also indicated in each panel.

Isojima et al. www.pnas.org/cgi/content/short/0908733106

8 of 12

Fig. S4. Effects of SP600125 and TG003 on mPER2 stabilization in 293T cells. (A) 293T cells cotransfected with Myc-tagged mPER2 (Myc-mPER2) and CKI␧(tau)were incubated with or without 100 ␮M SP600125 (Left) or TG003 (Right) for the indicated times, and the lysates were collected and analyzed by Western blotting.
Equal portions of three samples were mixed and analyzed in each lane. Myc-mPER2 was detected with an anti-Myc antibody (upper panels) and Tubulin was also
blotted as an internal control (lower panels). Note that the two compounds inhibited the mobility shift of mPER2 caused by the coexpression with CKI␧(tau). (B)Quantification of the results of (A). The densities of immunoreactive bands were measured, and the density of Myc-mPER2 was normalized to that of Tubulin
in the same sample (Myc-mPER2/tubulin). The graph represents the relative value of Myc-mPER2/Tubulin at the indicated incubation times with 100 ␮M SP600125(Left) or TG003 (Right) compared with the control (DMSO) samples.

Isojima et al. www.pnas.org/cgi/content/short/0908733106

9 of 12

Fig. S5.
CKI␧(tau) destabilizes the LUC::mPER2 and LUC::mPER1 proteins but not LUC or LUC::mBMAL1. (A) Effects of CKI␧(tau) on the stability ofLuciferase-fused clock proteins in 293T cells. The Luciferase activity in 293T cells transfected with the expression vector for LUC, LUC::mPER2, LUC::mPER1, or
LUC::mBMAL1, and CKI␧(tau) was measured 48 h after transfection. Note that the co-expression with CKI␧(tau) attenuated the bioluminescent activity of
LUC::mPER2 and LUC::mPER1, but not of LUC or LUC::mBMAL1. Each value represents the mean ⫹ SEM (n ⫽ 4). (B) Effects of CKI␧(tau) on the degradation ratesof Luciferase-fused clock proteins in 293T cells. The average ⫾ SEM. (n ⫽ 3) of the normalized bioluminescent activities of 293T cells cotransfected with LUC-fusedclock protein reporters (LUC::mPER2, LUC::mPER1, or LUC::mBMAL1) and CKI␧(tau) (circles) or control vector (squares) after treatment with CHX was plotted. The
fitted curve is also indicated in each panel.

Isojima et al. www.pnas.org/cgi/content/short/0908733106

10 of 12

Fig. S6. Temperature insensitivity of the CKI␧ phosphorylation activity. (A) Schematic diagram of PERIOD2 (PER2) and the sequence alignment around the
␤TrCP-binding (SSGYGS) and FASPS motif. Locations of the PAS domains (light blue), the ␤TrCP-binding motif (orange), the CKI-binding domain (red), and the
CRYPTOCHROME-binding domain (yellow) are indicated. Location corresponding to the amino acid sequence of the synthetic peptide substrate is indicated bybars above the diagram. Sequence corresponding to the ␤TrCP-peptide (amino acids 473–502, a serine-rich region around the ␤TrCP-binding motif in PER2) and
FASPS-peptide (amino acids 642– 666, a region around FASPS-related phosphorylation sites) are underlined in the aligned sequence. (B) The catalytic domains
of CKI␧ (wt) and CKI␧ (tau) [⌬CKI␧(tau) and ⌬CKI␧(wt)] and full-length CKI␧ were purified as described in the Materials and Methods, separated by electrophoresis,
and stained with Coomassie Brilliant Blue. (C) Autophosphorylation of CKI␧ before performing in vitro kinase assays. Dephosphorylated CKI␧ was incubated withor without ATP for 60 min. Note that CKI␧ was phosphorylated by the incubation with ATP (right-most lane). Lanes ‘‘M’’ in both (B) and (C) were standard
molecular weight markers. (D) Phosphorylation of FASPS-peptide by ⌬CKI␧(wt) (circles) and ⌬CKI␧(tau) (squares) was assayed at 25 °C (blue) and 35 °C (red). (Inset)
Expanded plot of the ⌬CKI␧(tau) phosphorylation activity. Assay conditions are described in the experimental procedures.

Isojima et al. www.pnas.org/cgi/content/short/0908733106

11 of 12Other Supporting Information Files
Table S1
Table S2
Table S3
Table S4

Isojima et al. www.pnas.org/cgi/content/short/0908733106

12 of 12