Supplemental Material

Items in Supplemental Materials and their relationship to main text and main figures

Figure S1 relates to Figure 1 and shows the phase relationship of different circadian

transcripts.

Figure S2 relates to the Result section Low-amplitude temperature fluctuations can

synchronize circadian gene expression over a wide temperature range and shows that within

the examined range of temperatures (30°C and 40°C) circadian clocks can be phase-entrained

to low amplitude temperature oscillations.

Figure S3 relates to Figure 5 and shows the phase relationship between the bioluminescence

cycles produced by different reporter genes in cells exposed to T-cycles with different period

lengths.

Figure S4 relates to Figures 5 and S3 and shows the behavior of luminescence cycles

entrained to a 12-hour T-cycle after release into a constant temperature

Figure S5 relates to Figure 5 and shows the phase relationship of various rhythmically

expressed transcripts in cells entrained to a very short T-cycle of only 10 hours.

Figure S6 relates to Figure 6 and shows the downregulation of HSF1 and HFS2 expression

by RNA interference using shRNAs.

Table 1 lists the DNA primers and Taqman probes used in quantitative RT-PCR experiments

  2

Legends to Supplemental Figures

Figure S1. The expression of various mRNAs in NIH3T3 cells exposed to simulated

body temperature rhythms.

(A) Real-time RT-qPCR (TaqMan) was used to quantify mRNAs in whole cell RNAs

prepared from NIH3T3 cells at four-hour intervals during the sixth day of temperature

entrainment (orange) or during the sixth day at constant temperature (blue). The primer- and

probe-sequences used for measuring Bmal1, Rev-erbα, Per2, Per3, Cry1 mRNAs and Hsp90

pre-mRNA levels are given in Supplemental Table 1. Two different biological samples per

time point were analyzed. For graphical purposes, the same values are plotted twice.

Bioluminescence profiles of NIH3T3/Bmal1-luc cells subjected to the same temperature

conditions (B) and corresponding temperature recordings (C) are shown for comparison.

Figure S2. Temperature cycles can synchronize circadian clocks within a broad

temperature range.

NIH3T3/Bmal1-luc cells whose circadian oscillators were transiently synchronized by a

serum shock are efficiently re-synchronized by temperature cycles with different magnitudes

but with the same relative amplitudes. Note the different phase relationships between the

peaks of Bmal1-luciferase expression and the peak of temperature (blue curves), which likely

reflects an over-compensation of period length at reduced temperatures. Indeed, in cells

exposed to the indicated constant temperatures, free-running periods shortened when the

incubation temperatures were lowered (grey curves). Thus, the shorter period length at lower

temperatures elicits phase advances under phase-entrained conditions. All bioluminescence

data were filtered by moving average transformation.

Figure S3. The phase relationship of circadian gene expression in cells exposed to T-

cycles of different lengths.

PER2::luciferase immortalized tail fibroblasts (turquoise), NIH3T3/DBP-luciferase

fibroblasts (pink), NIH3T3 fibroblasts transfected with a Bmal1-luciferase (blue), or a CMV-

luciferase reporter (grey, used as a non-circadian control) were exposed to temperature T-

cycles (35.5-38.5°C) with the period lengths indicated above the panels. Bioluminescence

data were filtered by moving average transformation on 24h intervals. For cells subjected to

temperature cycles of period lengths shorter than 24h, an enlarged panel on the right shows

  3

the 60 last hours of monitoring, whose bioluminescence data are filtered by moving average

transformation on 6, 10, 14 and 18h intervals respectively, allowing a better comparison of

the phases of each reporter. Note that under normal conditions Per2 and Dbp expression are

nearly antiphasic to Bmal1 expression.

Figure S4. Circadian oscillators of NIH3T3 cells entrained to 12-hour temperature T-

cycles revert to approximately 24-hour low amplitude cycles after release into constant

temperature.

(A). Left panel. NIH 3T3 cells transiently transfected with a pBmal1-luciferase (pBmal1-luc)

reporter plasmid or stably transfected with a Dbp-luciferase (Dbp-luc) reporter gene were

incubated with fresh medium and then immediately entrained to square-wave T-cycles of 12

hours (6 h 38°C/6h 34°C) during the time period indicated on the x-axis and then released to

a constant temperature of 34°C. Note that the bioluminescence cycles generated by the two

transgenes are almost antiphasic under both entrained and free-running conditions. Right

panel. Same as in left panel, but the T-cycles were initiated after a pre-run of six hours at

37°C after the medium change. (B) Same data as in (A), but shown individually for pBMAL1

and Dbp-luc, to illustrate the phase differences for the same reporters caused by the different

entrainment protocols (i.e. with or without a 6-hour pre-run). Note that the first valleys and

peaks at constant temperature are observed after 12 to 14 hours, before the oscillators resume

an approximately 24-hour cycle.

Figure S5. The expression of different genes in NIH3T3 cells exposed to temperature

square cycles of 10 hours.

(A) Programmed (blue) and measured (yellow and turquoise) temperature square cycles of

10h period (5h at 34°C, 5h at 39°C) used to entrain NIH3T3 fibroblasts. RNAs were prepared

from cells at two-hour intervals during the tenth and eleventh cycles (vertical grey arrows).

(B) Real-time RT-qPCR (SYBR Green) was used to quantify pre-mRNA of Hsp105, and

mRNAs of Per2, Bmal1, Rev-erbα and Dbp in whole cell RNAs prepared from these cells.

The primer-sequences used are given in Supplemental Table 1. Four biological samples per

time point were analyzed (each curve represents a pool of two biological samples).

  4

Figure S5. The accumulation of HSF1 and HSF2 mRNAs and proteins is down-

regulated in the presence of the corresponding shRNAs.

(A) Western-blots of total cellular proteins with antibodies against HSF1 and U2AF65

(loading control). Note the slower migration of HSF1 proteins upon a 2-hour heat shock at

42°C. The more slowly migrating proteins probably represent the hyperphosphorylated

(activated) forms of HSF1. Protein extracts were prepared form primary tail tip fibroblasts

(from wild-type and Hsf1 knockout mice) or from NIH3T3 fibroblasts transfected with

different shRNA vectors (pSUPER-empty, pshHSF1, pshHSF2 or pshHSF1+2) or

untransfected NIH3T3 fibroblasts (NT). (B) Western-blot of total cellular proteins with

antibodies against HSF2 and U2AF65 (loading control). (C) Real-time RT-PCR (TaqMan)

assays performed on mRNAs prepared from same cells that were used for the data presented

in (A) and (B), after a 2-hour heat shock at 42°C. The cells transfected with shRNA plasmids

also expressed green fluorescent protein (GFP) from a PGK promoter and could thus be

FACS-sorted before RNA and protein extracts were prepared. This ensured that only shRNA

expressing cells were included for the Western-blot and RT-qPCR experiments. The primer-

and probe-sequences used for measuring Hsf1 and Hsf2 mRNAs levels are given in

Supplemental Table 1.

  5

Supplemental Table 1. DNA sequences of primers and probes used for the real-time RT-

PCR experiments. TaqMan

mBmal1-F 5′-CCAAGAAAGTATGGACACAGACAAA-3′

mBmal1-R 5′-GCATTCTTGATCCTTCCTTGGT -3′

mBmal1 probe 5′-FAM-TGACCCTCATGGAAGGTTAGAATATGCAGAAC-TAMRA-3′

mRev-erbα-F 5`-TGAATGGCATGGTGCTACTG-3`

mRev-erbα-R 5`-CAGGCGTGCACTCCATAGT-3`

mRev-erbα probe 5`- FAM-GTAAGGTGTGTGGGGACGTG-TAMRA-3`

mPer2-F 5`-GTAGCGCCGCTGCCG-3`

mPer2-R 5`-GCGGTCACGTTTTCCACTATG-3`

mPer2 probe 5`-FAM-CGCGTCGCCCTCCGCTGT-TAMRA-3`

mPer3-F 5'-CCAGGTCACGTGGGACACA-3'

mPer3-R 5'-TCAATGACTGGCGTTTCAGAGT-3'

mPer3 probe 5'-FAM-AGCCGTGCGTTACCTCACCACAGTC-TAMRA-3'

mCry1-F 5'-CTGGCGTGGAAGTCATCGT-3'

mCry1-R 5'-CTGTCCGCCATTGAGTTCTATG-3'

mCry1 probe 5'-FAM-CGCATTTCACATACACTGTATGACCTGGACA-TAMRA-3'

pre-mHsp90-F 5'-CAGGACCTGGGCTAGGAAAT-3'

pre-mHsp90-R 5'-CAGCTAACCTTCCCCACAAA-3'

pre-mHsp90 probe 5'-FAM-AAGCTTTGGAGACCTTTGCA-TAMRA-3'

mHSF1-F 5'-GGACACAGACGCGCTCAT-3'

mHSF1-R 5'-CCTGGTCAAACACGTGGAAG-3'

mHSF1 probe 5'-FAM-GAGCCCGAGTGGGAACAG-TAMRA-3'

mHSF2-F 5'-GGCGAGCTTTGTGAGACAACT-3'

mHSF2-R 5'-CCATCTCTTTCCTGTTTGATAATTCC-3'

mHSF2 probe 5'-FAM-TGTATGGCTTCCGAAAAGTAGTGCATATCGAA-TAMRA-3'

GAPDH-F 5`-CATGGCCTTCCGTGTTCCTA-3`

GAPDH-R 5`-CCTGCTTCACCACCTTCTTGA-3`

GAPDH probe 5`-FAM-CCGCCTGGAGAAACCTGCCAAGTATG-TAMRA-3`

pre-m45S-F 5`-ACACCCGAAATACCGATACG-3'

pre-m45S-R 5'-TAGCTGCGTTCTTCATCGAC-3'

pre-m45S probe 5'-FAM-TCTTAGCGGTGGATCACTCG-TAMRA-3'

SYBR Green

mRps9-F 5′-GACCAGGAGCTAAAGTTGATTGGA-3′

mRps9-R 5′-TCTTGGCCAGGGTAAACTTGA-3′

mCyclophillin-F 5′-GGAGATGGCACAGGAGGAA-3′

mCyclophillin-R 5′-GCCCGTAGTGCTTCAGCTT-3′

pre-mHsp105-F 5′-GTGTCAGGGTCCTGTGGAGT-3′

pre-mHsp105-R 5′-GACCCCACCTCCTCAGTGTA-3′

mBmal1-F 5′-CCAAGAAAGTATGGACACAGACAAA-3′

mBmal1-R 5′-GCATTCTTGATCCTTCCTTGGT-3′

  6

mPer2-F 5′-ATGCTCGCCATCCACAAGA-3′

mPer2-R 5′-GCGGAATCGAATGGGAGAAT-3′

mDbp-F 5′-TGGCCCGAGTCTTTTTGC-3′

mDbp-R 5′-GCGTCCAGGTCCACGTATTC-3′

mRev-erbα-F 5′-TGAATGGCATGGTGCTACTG-3′

mRev-erbα-R 5′-CAGGCGTGCACTCCATAGT-3′

 

Saini_FigS1

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NIH3T3/Bmal1-luc cells

mCry1

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time (hours)

mPer3

126 130 134 138 142 146 126 130 134 138 142 146 126 time (hours)

mPer2

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mBmal1

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mpreHsp90

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126 130 134 138 142 146 150 154 158 162 166 170 174

constant 37°C temperature cycles (35.5-38.5°C)

time (hours) 126 130 134 138 142 146 150 154 158 162 166 170 174

constant 37°C temperature cycles (35.5-38.5°C)

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constant 38.9°C temperature cycles 37.3-40.5°C

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pCMV-luc pBmal1-luc Per2-luc

DBP-luc

6h period 10h period

14h period

24h period 30h period 34h period 40h period

18h period

temperature

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pBmal1-luc, 0 hours prerunDBP-luc, 0 hours prerun

pBmal1-luc, 0 hours prerunpBmal1-luc, 6 hours prerun

DBP-luc, 0 hours prerunDBP-luc, 6 hours prerun

pBmal1-luc, 6 hours prerunDBP-luc, 6 hours prerun

Saini_FigS5

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pool1 pool2 temperature

preHsp105

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Per2 Bmal1

Rev-erbα Dbp

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temperature program liquid probe 1 liquid probe 2

Saini_FigS6

Temperature °C WT

U2AF65

115 37 42

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48

HSF1 (~85 and 95 KDa)

Temperature °C

U2AF65

HSF2 (~70 KDa)

115 kDa

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HSF1 KO

37 42

3T3 NT

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emptyV

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