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Figure E1
PF670462 suppresses IgE-mediated degranulation in BMMCs generated from wild-type mice.
PF670462 or corticosterone (CORT) was added to the culture of BMMCs derived from C57BL6 mice at 72 hours after the media change for synchronization as described in Fig. 1A. Fifteen or 240 minutes after the addition of PF670462 or CORT, the wild-type BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in wild-type BMMCs pretreated with PF670462 or CORT for 15 or 240 minutes (n = 3). Values represent means±SD. *p0.05
Figure E2
PF670462 and corticosterone do not affect the viability of BMMCs.
Wild-type PER2LUC BMMCs (1 x 105 cells/well) were cultured with or without the indicated concentrations of PF670462 (A) or corticosterone (CORT) (B) for 24 hours in a 96-well microtiter plate. The cell viability was then determined by trypan blue dye exclusion(n = 3).Values represent means ± SD.
Figure E3
PF670462 and corticosterone do not affect the base-line cellular bioenergetics of BMMCs.
Wild-type BMMCs attached to a microtiter plate designed for the extracellular flux analyzer were cultured with or without the indicated concentrations of PF670462 or corticosterone (CORT) for 30 minutes. Oxygen consumption rate (OCR; an indicator of mitochondrial respiration) and extracellular acidification rate (ECAR) (an indicator of glycolysis) were measured at intervals of approximately 2-5 minutes. Plotted basal OCR and ECAR levels providing a snap-shot of the bioenergetics profiles of wild-type BMMCs treated or untreated with PF670462 or CORT are shown. There were no significant differences in OCR or ECAR among non-treated, vehicle-treated, PF670462- and CORT-treated BMMCs.
Figure E4
Wild-type BMMCs express CK1 protein
Cell lysates were obtained from 12-hour or 24-hour cultured (following a media change for synchronization) PER2LUC BMMCs with (WT) or without Clock mutation (ClockΔ19) and were subjected to Western blot analysis with anti-CK1 or anti--actin antibody.
Representative data are shown in the upper panel and the quantitative analysis (CK1/-actin ratio) is shown in the lower panel. Please note that there are comparable amounts of CK1 protein between wild-type and Clock-mutated PER2LUC BMMCs.
Figure E5
CK1/ inhibitor D4476suppresses IgE-mediated degranulation in mast cells associated with increased PER2 levels
A. PER2LUC bioluminescence of BMMCs derived from Per2Lucknock-in mice (PER2LUC BMMCs) was monitored after a media change for synchronization for 120 hours. Addition of D4476 (5 or 50 M) or corticosterone (CORT) (300 nM) at 72 hours following the media change (indicated by the arrows) increased PER2LUC levels in PER2LUC BMMCs 240 minutes after addition of D4476 and thereafter.
B. D4476 (5 or 50 M) or CORTwas added to PER2LUC BMMCs culture 72 hours after the media change as described in A. Fifteen or 240 minutes after the addition of D4476, PER2LUC BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in PER2LUC BMMCs (n = 3).
C.D4476 or CORT was added to PER2LUC BMMCs culture 72 hours after the media change as described in A. Fifteen or240 minutes after the addition of D4476, FcRI levels were evaluated by FACS analysis in PER2LUCBMMCs (n = 3).
MFI: mean fluorescence intensity.
D.PER2LUC BMMCs (1 x 105 cells/well) were cultured with or without the indicated concentrations of D4476 for 24 hours in a 96-well microtiter plate. The cell viability was then determined by trypan blue dye exclusion (n = 3).Please note that D4476 (10-50 M) did not affect the cellular viability.
Values represent means ± SD. *p0.05
Figure E6
PF670462 or corticosterone fails to suppress IgE-mediated degranulation in Per2-mutated mast cells.
A. PF670462 or corticosterone (CORT) was added to the culture of BMMCs derived from wild-type mice or mPer2m/m mice 72 hours after the media change for synchronization as described in Fig. 1A. Fifteen or 240 minutes after the addition of PF670462 or CORT, wild-type or Per2-mutated BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of-hexosaminidase was evaluated in wild-type or Per2-mutated BMMCs (n = 3).
B. PF670462 or CORT was added to wild-type or Per2-mutated BMMCs culture 72 hours after the media change as described in A. Fifteen or 240 minutes after the addition of PF670462 or CORT, FcRI levels were evaluated by FACS analysis in wild-type and Per2-mutated BMMCs (n = 3). MFI: mean fluorescence intensity.
Values represent means±SD. *p0.05
Figure E7
Specific CK1 inhibitor PF4800567 does not affect PER2 levels and IgE-mediated degranulation in mast cells.
A. PER2LUC bioluminescence of BMMCs derived from Per2Lucknock-in mice (PER2LUC BMMCs) was monitored after a media change for synchronization for 120 hours. Addition of PF4800567 (1 or 10 M) at 72 hours following the media change (indicated by the arrows) failed to increase PER2LUC levels in PER2LUC BMMCs. Corticosterone (CORT) was used as a positive control.
B. PF4800567 or CORT was added to PER2LUC BMMCs culture 72 hours after the media change as described in A. Two hundred forty minutes (4 hours) after the addition of PF4800567 or CORT, PER2LUC BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in PER2LUC BMMCs pretreated with PF670462 or CORT for 240 minutes.PF4800567 did not affect IgE-mediated release of -hexosaminidase (n = 3). Values represent means ± SD. *p < 0.05,
Figure E8
PF670462 or corticosterone suppresses IgE-mediated allergic reactions in IgE-sensitized mast cells as well as in unsensitized mast cells.
A. PER2LUC bioluminescence of IgE-sensitized or unsensitized BMMCs derived from Per2Lucknock-in mice (PER2LUC BMMCs) was monitored after a media change for synchronization for 120 hours. Addition of PF670462 (1 or 10 M) or corticosterone (CORT) (300 nM) at 72 hours following the media change (indicated by the arrows) significantly increased PER2LUC levels in both IgE-sensitized (IgE+) and unsensitized (IgE-) wild-type PER2LUC BMMCs 240 minutes after addition of the drugs and thereafter.
B. PF670462 or CORT was added to IgE-sensitizedwild-type PER2LUC BMMCs culture 72 hours after the media change as described in A. Two hundred forty minutes after the addition of PF670462 or CORT, IgE-sensitized PER2LUC BMMCs were stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in the IgE-sensitized wild-type PER2LUC BMMCs (n = 6).
C.PF670462 or CORT was added to IgE-sensitizedwild-type PER2LUC BMMCs culture 72 hours after the media change as described in A. Two hundred forty minutes after the addition of PF670462 or CORT, FcRI levels were evaluated by FACS analysis in IgE-sensitized wild-type PER2LUCBMMCs (n = 6). MFI: mean fluorescence intensity.
Values represent the means ± SD. *p0.05
Figure E9
Overexpression PER2 suppresses IgE-mediated degranulation and FcRI expression in mast cells.
A. Real-time PCR analysis for Per2 mRNA expression in mock- orPER2-overexpressing BMMCs using retroviral vector transfection. Please note that PER2-overexpressing BMMCs show abundant Per2 mRNA expression (n = 4).
B. Quantitative analysis of FcRI levels by FACS staining (MFI: mean fluorescence intensity) on mock or PER2 overexpressing BMMCs12 hours after a media change for synchronization (n = 4).
C. IgE-dependent intracellular Ca2+ mobilization in mock- orPER2-overexpressing BMMCs. Twelve hours after a media change for synchronization, the mock or PER2 overexpressing BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated intracellular Ca2+ mobilization was evaluated in the mock or PER2 overexpressing BMMCs (n = 6).
D. IgE-mediated release of -hexosaminidase from mock or PER2 overexpressing BMMCs. Twelve hours after a media change for synchronization, the mock or PER2 overexpressing BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in the mock or PER2 overexpressing BMMCs (n = 4).
E. IgE-mediated release of -hexosaminidase from wild-type (WT) or Per2-mutated BMMCs (mPer2m/m). Twelve hours after a media change for synchronization, the wild-type (WT) or Per2-mutated BMMCs derived from mPer2m/mwere sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in wild-type and Per2-mutated BMMCs (n = 6).
F. Quantitative analysis of FcRI levels by FACS staining (MFI: mean fluorescence intensity) on wild-type (WT) or Per2-mutated BMMCs derived from mPer2m/m12 hours after a media change for synchronization (n = 6).
Values represent the means ± SD. *p < 0.05
Figure E10
Aminophylline, a conventional bronchodilator for asthma, suppresses IgE-mediated degranulation and FcRI levels associated with increased PER2 levels in mast cells.
A. PER2LUC bioluminescence of BMMCs derived from Per2Lucknock-in mice with or without Clock-mutation (ClockΔ19) after a media change for synchronization for 120 hours (PER2LUC BMMCs). Addition of aminophylline (50 or 500 M) 72 hours following the media change (indicated by the arrow) increased PER2LUC levels in wild-type (WT), but not Clock-mutated (ClockΔ19), PER2LUC BMMCs. Corticosterone (CORT) (300 nM) was used as a positive control.
B. Aminophylline or CORT was added to wild-type or Clock-mutated PER2LUC BMMCs culture 72 hours after the media change as described in A. Two hundred forty minutes after the addition of aminophylline, PER2LUC BMMCs were sensitized with IgE and stimulated with anti-IgE antibody. Following the activation, IgE-mediated release of -hexosaminidase was evaluated in wild-type or Clock-mutated PER2LUC BMMCs pretreated with aminophylline for 240 minutes (n = 6).
C.Aminophylline or CORT was added to wild-type or Clock-mutated PER2LUC BMMCs culture 72 hours after the media change as described in A. Two hundred forty minutes after the addition of aminophylline, FcRI levels were evaluated by FACS analysis in wild-type and Clock-mutated PER2LUCBMMCs (n =3). MFI: mean fluorescence intensity.
Values represent the means ± SD.*p < 0.05
Figure E11
Kinetics of PER2LUC levels in mast cells in vivo
A. Representative pictures of in vivo imaging of mast cell-deficient W/Wv mice reconstituted with subcutaneous injections of BMMCs derived from Per2Lucknock-in mice with or without Clock mutation (ClockΔ19) at ZT4 (10:00 AM) or ZT16 (22:00 PM).
B.Quantitative analysis of the data in A (n = 10).
Values represent means ± SD.*p < 0.05
Figure E12
Comparable numbers of mast cells in the skin between mice reconstituted with wild-type BMMCs and Clock-mutated BMMCs.
A. Representative pictures of toluidin blue staining of mast cells (indicated by arrows) in the back skin from mast cell-deficient W/Wv mice reconstituted with subcutaneous injections (back skin) of BMMCs (1.5 x 106/mouse) derived from wild-type or ClockΔ19 mice. Scale bar represents 100 m.
B. Quantitative analysis of A (n = 4).
Values represent means ± SD.
Figure E13
OVA-induced nose scratching and sneezing in OVA-sensitized mice was mast cell-dependent.
Mast cell-deficientW/Wv mice were systemically reconstituted with wild-type BMMCs. The mice were then sensitized with OVA plus alum, followed by OVA or PBS challenge as described in Figure 4 and in the Methods.
A.The frequency of nasal rubs or sneezing was counted for a 10-minute period following the last intranasal challenge with OVA (n=5.)
B. OVA-specific IgE levels in the serum of mice following PBS or OVA challenge (PBS or OVA) evaluated by ELISA (n = 5).
Values represent means ± SD.*p < 0.05