Figure S1: PER Is Phosphorylated in Brains and DpN1 Cells
Protein samples were prepared from extracts collected at ZT4. Phosphatase (800 units) was incubated with each protein sample at 30 °C for 30 min. After, the samples were immediately mixed with 2X SDS-PAGE loading buffer, boiled for 5 min, analyzed by Western blot, and probed with PER-GP40. Sodium vanadate was used to block phosphatase activity.
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Figure S2: CRY1 and TIM Responses to Light after 48 h in DD
(A) Clock protein abundance in DpN1 cells changes in response to light after prolonged exposure to dark. DpN1 cells were cultured for 48 h under DD and then exposed to light for 540 min. Cells were collected at the designated times. Cell homogenates were analyzed by Western blot and probed for CRY1 (GP37), TIM (GP47), PER (GP40), and CRY2 (GP51) (upper panel). The time courses of declines were quantitated by chemiluminescence, and band intensity was normalized against α-tubulin (lower panel). Time 0 is before lights on.
(B) Effects of inhibitors on the light-induced decrease in CRY1 and TIM. Cells were pretreated with DMSO (the vehicle control, left), MG115 (final concentration of 40 μM in DMSO) for 2 h prior to light exposure (center), or GSK-3β inhibitor VIII (final concentration 20 μM in DMSO) for 2 h prior to light exposure (right). CRY1 abundance (GP37) and TIM abundance (GP47) were monitored by probing Western blots of cells collected at the designated times during the 120-min light exposure.
(C) CRY1 mediates light-induced TIM degradation in DpN1 cells. Cells were pretreated with dsRNA against GFP (left), cry1 (center), or tim (right) prior to light exposure. CRY1 abundance (GP37) and TIM abundance (GP47) were monitored by probing Western blots of cells collected at the designated times during the 120-min light exposure.
Results: We found a light-induced decrease in CRY1 in untreated DpN1 cells after culturing the cells for 48 h in DD. Once lights were turned on after 48 h in DD, there was a rapid decrease in CRY1 and TIM, followed by a slower decrease in PER, followed by a decrease in CRY2(A), similar to the temporal cascade of protein decrements found in . The delayed decrease in both PER and CRY2 abundance after light exposure was not due to accelerated protein synthesis, relative to CRY1 and TIM, because the same temporal sequence of declining protein accumulation was found following treatment of the cells with the protein synthesis inhibitor cycloheximide prior to light exposure (unpublished data).
The light-induced decrease in both CRY1 and TIM was blocked by the proteasome inhibitor MG115, showing that the decrease in CRY1 is mediated by proteosomal degradation (B, center), as occurs in Drosophila
]. The lack of light-induced decrease in TIM with MG115 treatment was also likely due to lack of proteasomal degradation of TIM itself, as the decrease in TIM by light is not necessarily accompanied by a decrease in CRY1. In fact, the GSK-3β inhibitor VIII blocked the light-induced decrease in CRY1, but did not
inhibit TIM's degradation by light (B, right). These data, along with the dsRNA data (C, center) show that CRY1 can mediate light-induced TIM degradation, with or without inducing its own degradation. The results further suggest the involvement of a GSK-3β-like kinase in the degradation of monarch CRY1 by light.
Using dsRNA, we showed that the light-induced decrease in TIM after 48 h in DD is also mediated through CRY1 (C). Pretreatment of cells with dsRNA targeting cry1 prior to turning the lights on caused a substantial (70%) reduction in CRY1 in darkness just prior to light exposure (time 0) and greatly reduced the decrease in TIM abundance in response to light, compared to controls (cells treated with dsRNA against GFP). Double stranded RNA targeting tim reduced TIM abundance prior to and throughout light exposure, but did not deter CRY1′s rapid decrease following lights on. Collectively, the data show that CRY1 mediates the light-induced decrease in TIM in DpN1 cells, with or without inducing CRY1′s own degradation.
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Figure S3: A Blue-Light Photoreceptor Entrains the Adult Eclosion Clock and Causes CRY1 and TIM Degradation in DpN1 Cells
(A) Experimental paradigm for adult eclosion studies. Top panel shows the wavelength and relative light intensities used. Lower panel depicts the timing of the three light pulses (white, blue, and orange) during the dark period prior to placement in constant darkness. Pupae were kept in 12-h-light: 12-h-dark (LD) conditions for 7 d at 21 °C in a Percival incubator. The incubator was then put into constant dark (DD). During first night of DD, a 1-h light pulse was given at ZT 21 using a white light arc lamp (66901, Newport Oriel Instrument) with either an orange 540-nm long-wavelength pass filter (E540, Gentex) or a blue 450-nm broadband interference filter (57541, Newport Oriel Instrument). Light profiles were measured with a USB2000 spectrophotometer (Ocean Optics). Animal eclosion was monitored by standard video surveillance equipment. The number of animals eclosed per hour was recorded.
(B) Eclosion profiles for all four groups (including “no-pulse” control) for each of the 3 d in constant darkness.
(C) Data from all 3 d in DD for each group pooled relative to circadian time.
(D) Light effects on CRY1 and TIM degradation in DpN1 cells. After 48 h of culture in DD, cells were either kept in the dark or exposed to white light, blue light, or orange light, using the light filters described above. Cell homogenates were analyzed by Western blot and probed for CRY1 (GP37) and TIM (GP47).
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Figure S4: Co-Localization of CRY1 and TIM in the PL
Double-labeling immunofluorescence of CRY1 (using CRY1-GP37, left column) and TIM (using TIM-R38, right column) are shown for two different cells in the PL (upper and lower rows). Only two of the four TIM-positive cells in the PL co-localized with CRY1, which was found in 6/6 brains examined.
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Figure S5: Distribution of TIM Immunoreactivity in Glomerular-Like Arborization and Adjacent Cells in the OL
(A) Schematic representation of a frontal section illustrating the topology of TIM-immunoreactive cells using antibody TIM-R38. RE, retina; LA, lamina; ME, medulla; LO, lobula; PL, pars lateralis; PI pars intercerebralis.
(B–D) Double-labeling of TIM (B) and CRY1 (C, using CRY1-GP37) staining in the glomerular-like arborization/cells in optic lobe (arrow). D is the merged image.
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Figure S6: A Light-Induced Decrease in CRY1 Is Not Essential for a Light-Induced Decrease in TIM in Either DpN1 Cells or Heads
This was shown in LD by giving a 1- hr light pulse from ZT 14–15 or from ZT 20–21, monitoring clock protein levels at the end of each light pulse and 3 h later, and comparing the levels with cells and heads kept in darkness (D); the formal properties of circadian clocks predict that light given early in the night (e.g., ZT 14–15) should delay the phase of the circadian clock oscillation, while light given late in the dark period (e.g., ZT 20–21) should advance the phase of the clock oscillation [44
(A) Paradigm for light pulse study. Arrows indicate collection times.
(B and C) Effects of lights pulse on CRY1 (GP37) and TIM (GP47) levels in DpN1 cells (B) and heads (C). Protein levels were determined by Western blots. Band intensity was quantified by chemiluminescence, and the values were normalized against α-tubulin. For each timepoint, samples collected in the dark (gray bars) are plotted next to samples collected after a light pulse (red and blue bars). Each bar is the mean ± SEM of three experiments.
Results: When a 1-h light pulse was given from ZT 14–15, TIM levels in both DpN1 cells and heads were significantly decreased, as expected, just after the light pulse (ZT 15), and the decrease was still present 3 hrs later (ZT 18) (B and C). However, there was no decrease in CRY1 abundance at either time point. Similar responses were seen in both DpN1 cells and heads when the light pulse was given from ZT 20–21 (B and C). In this instance, there was a small, but significant decrease in CRY1 3 h after lights off (ZT 0) in DpN1 cells (B).
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Figure S7: Monarch PER Alone or in Combination with Submaximal Inhibitory Doses of CRY2 (A) or with TIM (B) Does Not Repress dpCLK:dpCYC–Mediated Transcription Using Luciferase Reporter Gene Assays
The monarch butterfly per E box enhancer luciferase reporter (dpPer4Ep-Luc; 50 ng) was used in the presence (+) or absence (–) of monarch CLK/CYC expression plasmids (50 ng each). Monarch cry2 (5 and 15 ng), per (5, 15, and 50 ng) or tim (5, 15, and 50 ng) was used. Luciferase activity relative to β-galactosidase activity was computed. Each value is the mean ± SEM of three independent transfections. Western blot of FLAG-epitope-tagged protein expression levels for each concentration of each construct is depicted below the graph in (A).
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Figure S8: Effect of dsRNA against per on cry2 RNA Levels
DpN1 cells were treated with either dsRNA against GFP (ds GFP) or dsRNA against per (ds per). PER and CRY2 levels were assessed by Western blot analysis, using PER-GP40 and CRY2-GP51 (upper panel). Blots were imaged by chemiluminescences, and band intensity was quantified. The results were normalized against α-tubulin. Corresponding RNA levels for cry2 were assessed by qPCR (lower panel). The cry2 RNA values are expressed relative to the value with ds GFP treatment (100%). Each value is the mean ± SEM of three experiments.
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Figure S9: CRY2 Protein Levels in DpN1 Cells
(A) Verification of specific knockdown of CRY2 in D by dsRNA against cry2 (lower blots) compared with CRY2 abundance when treated with dsRNA against GFP (upper blots) at two time points (ZT 4 and ZT 12) over the 24-h period of study. CRY2-GP51 was used.
(B) Subcellular location of CRY2 in DpN1 cells. Photomicrographs depict CRY2 in nucleus only (left column) and in both nucleus and cytoplasm (right column). Upper row, CRY2 staining (CRY2-GP51); middle row, nuclear staining with SYTOX Blue; lower row, merged images.
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Figure S10: CRY2 RNA Distribution in Monarch Brain
(A) Schematic representation of a frontal section illustrating the topology of CRY2 RNA expression. RE, retina; LA, lamina; ME, medulla; LO, lobula; PL, pars lateralis; PI pars intercerebralis, SOG, suboesophageal ganglion.
(B) CRY2 RNA staining in a group of neurosecretory cells in pars intercerebralis (PI).
(C) CRY2 RNA staining in cells in pars lateralis (PL).
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Figure S11: Co-Localization of CRY2 and TIM in the PL
Double-labeling immunofluorescence of CRY2 (using CRY2-R42, left column) and TIM (using TIM-GP47, right column) are shown for a cell in the PL at ZT 18 (upper), ZT 21 (middle), and ZT 0 (lower). All four CRY2-positive cells in the PL colocalized with TIM, which was found in 4/4 brains examined.
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Figure S12: The Nuclei of PL Cells and CRY2 Staining
(A) Photomicrograph of a region near the PL stained with the nuclear stain propidium iodide. Arrows denote patchy nuclear staining in two CRY2-positive cells (two arrows for each cell), whereas arrowheads denote intense nuclear staining in surrounding cells.
(B) Nuclear CRY2 is co-localized with chromatin in the PL. The section (5 μm) was taken from a brain collected at ZT4. The section was stained for CRY2 (CRY2-R42; left) and counterstained with propidium iodide (middle); the staining in cytoplasm is due to overexposure to amplify the low intensity of nuclear staining. The merged image (right) shows co-localization (arrows)
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Figure S13: CRY2 Staining in Monarch Brain Using Antibodies R42 and GP51
(A and B) Double-labeling immunofluorescence of CRY2 staining in three cells in the PL using R42 (A) and GP51 (B). The fourth cell was out of the plane of section.
(C and D) Double-labeling immunofluorescence of CRY2 staining in a cell in the PI using R42 (C) and GP51 (D). All CRY2 positive cells in PI were co-localized with the two antibodies.
(E and F) CRY2 fluorescence in lower division of the central body (CB) using either R42 (E) or GP51 (F).
(G and H) CRY2 DAB staining in upper and lower subdivisions of the CB using either R42 (G) or GP51 (H).
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Table S1: Monarch Clock Genes Expressed in DpN1 Cell Line
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Table S2: Degenerate Primer Sequences
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The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/
) accession numbers for the monarch genes discussed in this article are period
(DQ184682), casein kinase II
α (EF554579), casein kinase II
β (EF554578), shaggy
(EF649714), and slimb