0 1 2 3 4 5
***
Corticoid excretion (ng/4h)
Hours after "lights off"
6 10 14 18 22 26 WT
KO
Supplemental Figure 1. 24-hour profile of corticoid excretion in fecal samples from WT and KO mice on the first day in DD (n = 5–6). *** P < .001 (2-way ANOVA with Bonferroni post-hoc test).
Leliavski et al. “Impaired stress response in Arntl−/− mice” - Supplemental data
WT KO
0.0 0.1 0.2 0.3 0.4
Adrenal weight/body weight, 10-3
0 1 2 3
Cortex-to-medulla ratio
WT KO
**
A B
Supplemental Figure 2. Anatomical examination of Arntl-deficient adrenal glands. (A) Relative adrenal weight in WT and KO mice (n = 9–13). (B) Adrenal cortex-to-medulla ratio in KO mice and age-matched WT controls (n = 5–6).
1
Supplemental Figure 3. Body axis angle (A) and submersion in water (B)
of WT and KO mice during immobility periods in the FST. (C) Rectal temperature measured in WT and KO mice at ZT12. * P < .05 (Student's t-test).
Supplemental Figure 4. Schematic representation of tested genes involved in
regulation of adrenocortical functions. Rhythmically expressed genes are labeled in red.
2
WT KO
0 10 20 30 40
Body angle (degrees)
0.0 0.1 0.2 0.3 0.4 0.5
Depth of submersion / body length
34 35 36 37 38
Rectal temperature (degrees C)
WT KO
WT KO
*
A B C
Cyp11a1 Cyp11b1 Hsd3b1 Por
corticosterone
mitochondria adrenocortical cell
Steroidogenesis Cholesterol
transport StarStard4 LdlrScarb1
ACTH
ACTH receptor signaling
Mc2rMrap Prkce
Transcriptional regulation Nr5a1
Nr0b1 Sp1 Cholesterol
uptake
Nr1h3
Supplemental Figure 5. mRNA levels of genes of interest in liver, epididymal adipose tissue and skeletal muscle from WT and KO mice at 48 h after “lights off” (n = 3–4). * P < .05, ** P < .01, *** P < .001 (Student's t-test).
3
***
Relative mRNA expressionRelative mRNA expressionRelative mRNA expression
Ldlr Por Stard4 Liver
Adipose
Muscle
0 Star 1 2 3 4
0.0 0.5 1.0 1.5 2.0
0.0 0.5 1.0 1.5
Ldlr Por Stard4 Star
Ldlr Por Stard4 Star
** *
* * *
Supplemental Table 1. Primer sequences used for quantitative RT-PCR
Gene Forward primer (5’ to 3’) Reverse primer (5’ to 3’)
Star TTGGGCATACTCAACAACCA GAAACACCTTGCCCACATCT
Mc2r AAGCCTCGTGGCAGTTTTGAA AGGATGAACATGCAGTCAATGAT
Ldlr TGACTCAGACGAACAAGGCTG ATCTAGGCAATCTCGGTCTCC
Prkce GGGGTGTCATAGGAAAACAGG GACGCTGAACCGTTGGGAG
Por ATGGGGGACTCTCACGAAGAC TCTTGCTGAACTCCGGTATCTC
Stard4 TGTTTGGTATGGAGAGTGTGGA GTCACAGCAGAGACTGACATTG
Scarb1 TTTGGAGTGGTAGTAAAAAGGGC TGACATCAGGGACTCAGAGTAG
Nr5a1 GTAACTGAACTGGTCAAACCCC AAGCCTGGCTAGTGCATGTC
Nr0b1 CGGATGATGCAGAGAGAGTACC AATGATGGGCCTGAAAAAGAGTT
Sp1 GCCGCCTTTTCTCAGACTC TTGGGTGACTCAATTCTGCTG
Nr1h3 CTCAATGCCTGATGTTTCTCCT TCCAACCCTATCCCTAAAGCAA
Cyp11a1 AGGTCCTTCAATGAGATCCCTT TCCCTGTAAATGGGGCCATAC
Cyp11b1 CTGAACCCAAATGTTCTGTCACC CAAAGTCCCTTGCTATCCCATC
Hsd3b1 AGCATCCAGACACTCTCATC GGAGCTGGTATGATATAGGGTA
Mrap ACTGTCATGGCCAACGG AGTGTGAGGCCAGCTGAT
Eef1a1 TGCCCCAGGACACAGAGACTTCA AATTCACCAACACCAGCAGCAA
4
