In vitro kinase assay
The inhibitory effects of TG003 and IC261 against CK1 isoforms were tested using the QuickScout screening assist mobility shift assay with the ATP concentration at the Km (4.1 μM for CK1α, 6.3 μM for CK1γ1, 10 μM for CK1γ2, 3.2 μM for CK1γ3, 7.7 μM for CK1δ, and 16 μM for CK1ϵ; Carna Biosciences, Inc., Kobe, Japan). Detailed information on the assay condition is available on the website of Carna Biosciences (http://www.carnabio.com). Full-length human CK1α, CK1γ1, CK1γ2, CK1γ3 and catalytic domain of human CK1ϵ were expressed as N-terminal GST-fusion protein using baculovirus system, and purified by using glutathione sepharose chromatography. Catalytic domain of CK1δ was expressed as N-terminal GST-fusion protein in E. coli, and purified by using glutathione sepharose chromatography.
PCR-amplified fragments of mCherry (Clontech) and PER3 (Accession: NP_058515) were fused in-frame by overlap-extension PCR method to generate mCherry-PER3, respectively, as described previously
 with some modifications. The combined fragment was inserted into pCAGIPuro vector, an IRES-based bicistronic expression vector where the gene of interest and a puromycin resistant gene are expressed from a single mRNA, which enables almost all of the cells selected with puromycin to express the gene product. PCR-amplified fragments of FLAG-tagged CK 1δ (Accession: BC015775) and ϵ (Accession: BC006490) were fused in-frame to the amino-terminus of EGFP via F2A peptide sequence by overlap-extension PCR method, which enables bicistronic expression of FLAG-tagged CK1 isoforms and EGFP. The combined fragments were inserted into pcDNA5/FRT/TO (Life Technologies). The reconstituted vector sequences are available upon request.
Cell culture and transfection
Flp-In/T-REx HEK293 cell (Life Technologies) was maintained in low glucose Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan), 100 units/ml of penicillin and 100 μg/ml of streptomycin (Nacalai Tesque). Cells were transfected with plasmid DNAs using polyethylenimine MAX (Polysciences) as described previously
, and then selected with hygromycin B (Life Technologies) for pcDNA5/FRT/TO vectors and puromycin (Nacalai Tesque) for pCAGIPuro vectors to establish the stable cell lines.
PER3 nuclear translocation assay
HEK293 cells seeded in a density of 1 × 105 cells/dish in polyethyleneimine-coated 35 mm glass bottom dishes (MatTek, Ashland, MA) were cultured for 2 days. Cells were pre-incubated with 0.1% dimethyl sulfoxide (DMSO) containing 30 μM TG003, 30 μM TG001, or 1 μM PF-670462 (Merck, Darmstadt, Germany) for 1 hour at 37°C before expression of CK1δ or CK1ϵ was induced with 1 μg/ml of doxycycline. After 8 hour incubation with doxycycline at 37°C, cells were fixed with 10% Formaldehyde Neutral Buffer Solution (Nacalai Tesque) for 10 min at room temperature. Cells were washed twice with PBS and then stained with 5 μg/ml of Hoechst33342 (Dojindo, Kumamoto, Japan) in PBS for 30 min at room temperature. The Hoechst33342 solution was removed and cells were washed with PBS, and stored in 1.5 ml PBS at 4°C in the dark until taking fluorescent images on the Confocal Laser Scanning Biological Microscope FV10i (Olympus, Tokyo, Japan).
The fluorescent images were analyzed by the compartmental analysis algorithm predefined in Cellomics BioApplications (Thermo Fisher Scientific, Waltham, MA). The nuclear-cytoplasmic ratio of the mCherry-PER3 signal intensity was quantified by dividing the mean average mCherry intensity in the nuclear area defined as “circ” by the mean average mCherry intensity of a “ring” around this area, which covered a cytoplasmic region. The distance of the circ to the nuclear outline was 16 pixels. The ring had a width of 4 pixels and a distance of 1 pixel from the nuclear outline. The fluorescent image containing over 15 objects (cells) counted by the compartmental analysis algorithm was used for analysis. The objects that were under 650 of the mean average EGFP intensity in the nuclear area were excluded. Analysis data was exported into Excel file for statistical analysis.
Male C57BL/6 J mice (5 weeks old) were purchased from Clea Japan, Inc. (Tokyo, Japan) and housed under controlled temperature (24 ± 1°C) and humidity (55 ± 10%) with a 12-hour light-dark cycle with food and water freely available. The animal experiments were approved by the Animal Care Committees of Tokyo Medical and Dental University (approval No. 0090173) and Kagoshima University (approval No. MD10053), and were conducted in accordance with the ethical guidelines for the study of experimental pain in conscious animals published by the International Association of the Study of Pain (1995)
 and the European Communities Council Directive of 24 November, 1986 (86/609/EEC).
Animal models and behavioral studies
To produce acute and persistent inflammatory pain, carrageenan (2% lambda carrageenan in saline, 25 μl, Sigma, St. Louis, MO) and complete Freund’s adjuvant (CFA, 25 μl, Sigma) were injected into the plantar surface of the right hindpaw under light halothane anesthesia, respectively
[46–49]. Control mice were treated with saline or incomplete Freund’s adjuvant (IFA, Sigma), respectively. Mechanical allodynia and thermal hyperalgesia were measured using the Dynamic Plantar Aesthesiometer (Ugo Basile, Comerio VA, Italy) and the Paw Thermal Stimulator (UCSD, San Diego, CA, USA), respectively as described
. In CFA model, these behavioral experiments were conducted 3 days after the injection. Intrathecal (i.t.) injection was given in a volume of 5 μl by percutaneous puncture through an intervertebral space at the level of the 5th or 6th lumbar vertebra, according to a previously reported procedure
[12, 50]. An investigator, who was unaware of the drug treatment, performed all of the behavioral experiments.
Six hours after carrageenan or saline injection, and 3 days after CFA or IFA injection, mice were anesthetized with sodium pentobarbital (50 mg/kg), and the lumbar spinal cord and DRGs (L4-L6) were quickly removed. Each sample was homogenized in a lysis buffer [150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1:100 diluted protease inhibitor cocktail (Sigma), and 50 mM Tris-HCl, pH 8.0]. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Proteins were separated by SDS-PAGE (7.5% gel) and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). Anti-CK1α (rabbit polyclonal, raised against amino acids 281-337 at the C-terminus of human CK1α; 1: 2,000; no. sc-28886, Santa Cruz Biotechnology, Santa Cruz, CA), anti-CK1ϵ (rabbit polyclonal, raised against amino acids 301-360 near the C-terminus of human CK1ϵ; 1: 1,000; no. sc-25423, Santa Cruz Biotechnology) and anti-CK1δ antibody (rabbit polyclonal, NC10, 1:4,000; kindly donated by Prof. Uwe Knippschild, Univ. Ulm, Germany) were used. The specificities of the three antibodies were characterized and reported previously in several studies including ours
[12, 41, 42, 51]. We have also conducted control staining experiments; omission of primary antibody or secondary antibody, and substitution of primary antibody with normal rabbit IgG. We did not obtain any signals from these control experiments (data not shown).
Immunoreactivity was detected by using the ECL system (GE Healthcare, Buckinghamshire, UK). An anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (mouse monoclonal, 1:20,000; no. MAB374, Chemicon, Temecula, CA) or β-actin (mouse monoclonal, 1:1,000; no. sc-47778, Santa Cruz Biotechnology) were used to normalize protein loading. Relative intensities of the bands were quantified by using an image analysis system with Image J software, version 1.40 g (National Institutes of Health, Bethesda, MD). At least two independent immunoblot experiments of three independent spinal cord and DRG samples were analyzed.
Patch-clamp recordings from spinal dorsal horn neurons
Adult mouse spinal cord slices were prepared according to the method of Yoshimura & Jessell
[21, 22]. Briefly, 6 hours after carrageenan or saline injection, and 3 days after CFA or IFA injection, transverse slices (thickness, 800-900 μm) of the L5 spinal segments with the L5 dorsal root attached were cut on a vibrating blade microtome. The slices were superfused with Krebs solution (10-15 ml/min) saturated with 95% O2 and 5% CO2 at 36 ± 1°C. The composition of Krebs solution was as follows (in mM): NaCl 117; KCl 3.6; NaHCO3 25; NaH2PO4 1.2; CaCl2 2.5; MgCl2 1.2, and glucose 11 (pH 7.4 after gas saturation).
Blind whole-cell patch-clamp recordings were made from the lamina II (substantia gelatinosa: SG) neurons ipsilateral to carrageenan, CFA, or vehicle (saline or IFA) injection in voltage clamp mode. Patch pipettes were fabricated from thin-walled, borosilicate, glass-capillary tubing (1.5 mm o.d., World Precision Instruments). After establishing the whole-cell configuration, neurons were held at the potential of -70 mV to record spontaneous excitatory postsynaptic currents (sEPSCs) and at the potential of 0 mV to record spontaneous inhibitory postsynaptic currents (sIPSC). Under these conditions, GABA- and glycine-mediated IPSCs and glutamate-mediated EPSCs, respectively, were negligible, because these holding potential were close to the reversal potentials of IPSCs and EPSCs, respectively
. Recording electrodes were filled with either potassium gluconate-based solution (in mM: K-gluconate 135; KCl 5; CaCl2 0.5; MgCl2 2; EGTA 5; HEPES 5; ATP-Mg 5; adjusted with KOH to pH 7.2) to investigate EPSCs, or Cs-based solution (in mM: Cs2SO4 110; tetraethylammonium 5; CaCl2 0.5; MgCl2 2; EGTA 5; HEPES 5; ATP-Mg 5; adjusted with CsOH to pH 7.2) to examine IPSCs. The resistance of a typical patch pipette is 5-10 MΩ. Membrane currents were amplified with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) in voltage-clamp mode. Signals were low-pass filtered at 5 kHz and digitized at 333 kHz with an A/D converter (Digidata 1322, Molecular Devices). Data were stored with a personal computer using pCLAMP10 software and analyzed with Mini Analysis software (Synaptosoft Inc., Decatur, GA, USA).
The average values of both frequency and amplitude of sEPSCs or sIPSCs during the control (1 min) and 5-10 min after the drug application (1 min period after the attainment of steady effect of each drug) were calculated and quantified as relative changes in frequency and amplitude. Since the characteristics of sEPSCs and sIPSCs parameters (frequency and amplitude) were not significantly different among naïve-, saline- and IFA-control, data from each control were combined.
IC261 was from Calbiochem, LaJolla, CA, USA. PF-670462 was obtained from Tocris bioscience, Bristol, UK. TG003 and TG001 were synthesized according the procedures described previously
. These drugs were made up as concentrated stock solution in DMSO, aliquoted and stored at –20°C. An aliquot was diluted to the desired concentration in saline or Krebs solution immediately prior to use. The dose ranges of IC261 and TG003 used were determined according to our previous report (for IC261)
 and preliminary study (for TG003).
Experimental data are expressed as mean ± SEM. Single comparisons were made using Student’s two-tailed paired or unpaired t-test. One-way ANOVA followed by the Dunnett’s or Tukey’s test was used for multiple comparisons. P < 0.05 was considered statistically significant.