Supplementary Figure 1. Effect of PKA stimulation on functional adrenal differentiation. A- Dexamethasone and ACTH treatments modulate Akr1b7 expression. Wild-type C57Bl/6 mice were treated for 5 days with vehicle (Ctrl) or Dexamethasone (Dex) or for 3 days with dexamethasone followed by two days of Synacthene retard (Dex + ACTH). Akr1b7 expression levels were then analysed by RTqPCR on mRNAs extracted from adrenals. Bars represent mean expression in 4 animals per group + SEM. B- ACTH treatment inhibits activity of the WNT pathway reporter TopGal. Transgenic mice bearing the WNT reporter gene TopGal were treated for three days with vehicle (Ctrl) or ACTH (synacthene retard) and reporter activity was detected by X-Gal staining. Inset shows a high magnification picture of ZG. C- ACTH treatment increases AKR1B7 staining area in the adrenal cortex of wild-type mice. Wild-type C57Bl/6 mice were treated for 5 days with vehicle (Ctrl) or Dexamethasone (Dex) or for 3 days with dexamethasone followed by two days of Synacthene retard (Dex + ACTH). AKR1B7 expression was detected by immunohistochemistry and stained areas were measured and expressed as a percentage of total adrenal cortex surface. Bars represent the mean of 4 adrenals per treatment group + SEM. D- Sf1:Cre induces LoxP recombination in all cortical cells. Sf1:Cre mice were mated with mT/mG reporter mice to generate Sf1:Cre;mTmG offspring. Recombination efficiency was analysed by immunodetection of GFP expression. Sections were also stained with Cyp11b2 (ZG) and Hoechst (nuclei). Recombination was induced in all cortical cells including ZG (inset). E- Prkar1a expression is decreased in Sf1:Cre; Prkar1a Fl/Fl mice. Prkar1a expression levels were analysed by RTqPCR on mRNAs extracted from wild-type and Sf1:Cre;Prkar1aFl/Fl adrenals. F-G-H Prkar1a ablation induces expression of Akr1b7, StAR and 20&HSD. Akr1b7, StAR and 20aHSD expression levels were analysed as above. I- 20&HSD expression domain is expanded in Sf1:Cre Prkar1aFl/Fl mice. 20aHSD expression was analysed by immunohistochemistry in 2.5 month-old nulliparous wild-type and Sf1:Cre;Prkar1aFl/Fl female mice. J- Akr1b7 staining area is increased in the adrenal cortex of Sf1:Cre;Prkar1a Fl/Fl mice. AKR1B7 staining in wild-type and Sf1:Cre;Prkar1aFl/Fl mice was evaluated as in C. K- Intra-adrenal aldosterone concentration is reduced in Sf1:Cre;Prkar1aFl/Fl mice. Intra-adrenal aldosterone concentration was evaluated by ELISA after steroids extraction from whole adrenal gland tissue. In E-H and I, bars represent mean expression in 6 animals per group + SEM. Statistical analyses were conducted by one-way ANOVA followed by Tukey's post-hoc test (A,C) or Student's t test (E-H, J-K). * p<0.05; ** p<0.005; *** p<0.0005. Scale bars represent 100 um. ZG, zona glomerulosa; ZF, zona fasciculata; M, medulla.
A
Akr1b7
15-
Relative mRNA quantification
B
C
Akr1b7 surface
D
Ctrl
ACTH (3 days)
*
TCF:LacZ
Ratio to whole
GFP/Hoechst/Cyp11b2
Sf1:Cre; mTmG
10
1.0
ZF
ZG
ZF
ZG
ZF
ZG
0.8
M
ZG
5
ZG
cortex
0.6.
M
M
‘ZG
Ctrl
0.4-
Ctrl
Dex
0.2-
Dex
0
Dex + ACTH
0-
Dex + ACTH
E
Prkar1a
F
Akr1b7
G
StAR
H
20aHSD
105
Relative mRNA quantification
Relative mRNA quantification
Relative mRNA quantification
Relative mRNA quantification
*
1.5
8
2.5
10
I
WT
Sf1:Cre; Prkar1a Fl/Fl
*
6
2.0
10
1.0
1.5
10
20a HSD
ZG
ZF
ZG
4
T
.
0.5
1.0
10
1
M
2
0.5
T
0
0
0
10”
J
Akr1b7 surface
Akr1b7 surface
K
Aldosterone
1.0-
150000
*
Ratio to whole
0.8-
cortex
0.6
T
pg/mg
100000
0.4
50000
I
0.2
WT
WT
0-
Sf1:Cre; Prkarta FI/FI
0
Sf1:Cre; Prkarta FI/FI
A
StAR
Relative mRNA quantification
5
4
3
NS
2
NS
1
0
0
1
2
4
6
8
24 hours Fsk
H295R cells
Supplementary Figure 2. Effect of PKA stimulation on WNT pathway phosphorylations. A- StAR expression is induced by forskolin in H295R cells. Human H295R cells were treated with either vehicle (0) or Forskolin 10-5M (Fsk) for 1 to 24 h and StAR expression was analysed by RTqPCR. Bars represent mean expression in at least three independent experiments (in triplicate) + SEM. B-O- Quantification of WNT pathway regulators phosphorylation in COS7 cells (B-H) and H295R cells (I-O). Western-blot signals presented in Fig. 2C were quantified and accumulation of phospho- proteins was normalised with accumulation of corresponding total proteins. Bars represent the mean of fold activation over DMSO (at least 4 independent experiments) for each time point + SEM. In all experiments, statistical analyses were conducted by one-way ANOVA followed by Dunett's post-hoc test. * p<0.05; ** p<0.005; *** p<0.0005; ns: Not significant.
B
pS552 b-catenin/b-catenin
C
pS675 b-catenin/b-catenin
D
ABC b-catenin/b-catenin
E
pT41-S45 b-catenin/b-catenin
DE
ns
-
*
*
Relative protein quantification
3
Relative protein quantification
2.0
Relative protein quantification
4
ns
15
Relative protein quantification
2.5
ns
ns
ns
DE
ns
1.5
ns
3
2.0
2
ns
r
1.5
1.0
2
1.0
1
0.5
1
0.5
0
0
0
0
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24 hours Fsk
COS7
COS7
COS7
COS7
F pS33-S37-T41 b-catenin/b-catenin
G
pS9 GSK3b/GSK3b
H
pY216 GSK3b/GSK3b
ns
ns
Relative protein quantification
1.5
Relative protein quantification
2.0
Relative protein quantification
2.0
ns
1.5
1.5
1.0
T
0
ns
ns
ns
1.0
1.0
0.5
T
0.5
0.5
0
0
0
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24
hours Fsk
0
1
2
4
6
8
24
hours F
Fsk
COS7
COS7
COS7
I
pS552 b-catenin/b-catenin
J
pS675 b-catenin/b-catenin
K
ABC b-catenin/b-catenin
L
pT41-S45 b-catenin/b-catenin
*
ns
4
3
2.0
C
Relative protein quantification
A
Relative protein quantification
Relative protein quantification
15
ns
Relative protein quantification
15
*
-
*
ns
ne
ns
3
ne
1.5
=
2
10
2
1.0
T
T
1
5
1
0.5
0
0
0
0
0
1
2
4
6
8
24
4 hours Fsk
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24
hours Fsk
0
1
2
4
6
8
24 hours Fsk
H295R
H295R
H295R
H295R
M pS33-S37-T41 b-catenin/b-catenin
N
pS9 GSK3b/GSK3b
O
pY216 GSK3b/GSK3b
ns
ns
DE
Relative protein quantification
2.0
05
2.5
1
ns
ns
Relative protein quantification
ns
Relative protein quantification
2.0
NS
A
ns
r
1.5
2.0
n
1.5
1.5
1.0
T
1.0
T
1.0
T
0.5
0.5
0.5
0
0
0
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24 hours Fsk
0
1
2
4
6
8
24
hours Fsk
H295R
H295R
H295R
WNT pathway phosphorylation in vivo
WT
Sf1:Cre; Prakria Fİ/FI
b-catenin
PS552 PS675 ABC
Activating
pT41/S45
Inactivating
Total
GSK3b
PS9
Inactivating
PY216
Activating
Total
GAPDH
Supplementary Figure 3. Phosphorylation of ß-catenin and GSK3B in response to constitutive PKA activation in
vivo. Activating and inactivating phosphorylations of ß-catenin and GSK3ß were analysed in adrenal protein extracts
from 5 wild-type and 5 Sf1:Cre; Prkar1a Fl/Fl mice, independent from samples shown in main manuscript Fig. 2D.
Supplementary Figure 4. Analysis of selected WNT pathway regulators expression in Sf1:Cre; Prkar1a Fl/Fl mice by RTqPCR. A- Heatmap representation of RTqPCR data (Log2) in 6 wild-type and 6 Sf1:Cre; Prkara1a Fl/Fl mice (independent from the animals used in Fig 3A). B- Representation of RTqPCR data as barplots, following conversion of Log2 values to linear values (2^-44Ct). C- Correlogram generated from RTqPCR data, showing Pearson's correlation coefficients between the 15 WNT pathway genes. Statistical analyses in B were performed by Student's t test. * p<0.05; ** p<0.005; *** p<0.0005; ns: Not significant.
A
Ndrg1
Wnt5a
Cdc73
Rnf43
Sf1:Cre;
Apc
Lrp5
Fzd7
Dixdc1
FrzB
Dab2
Wnt4
CyID
Znrf3
WT
Axin2
Prkar1a Fl/Fl
Log2 FC
C
Lef1
FrzB
-4.31
Apc
1
Dab2
-2.79
Dixdc1
-2.38
Ndrg1
0.8
Lef1
-2.27
Wnt5a
0.6
Wnt4
-2.14
Cdc73
Rnf43
-1.76
Rnf43
0.4
Axin2
-1.03
Lrp5
0.2
Fzd7
-0.90
Fzd7
Pearson’s r
Znrf3
-0,70
Dixdc1
0
CylD
-0,35
FrzB
Lrp5
0.45
Dab2
-0.2
Wnt5a
0.54
Wnt4
-0.4
Apc
0.84
Ndrg1
Lef1
1.10
Cdc73
1.44
Axin2
-0.6
CylD
-0.8
-4.5
3.0
Znrf3
median-centered DCt
-1
B
Relative mRNA quantification
FrzB
Wnt4
Dab2
Lef1
Rnf43
Fzd7
Znrf3
Axin2
1.5
1.5
1.5
1.5
1.0-
1.5
1.5
1.5
*
0.8
*
1.0
1.0
1.0
1.0
0.6
1.0
T
1.0
1.0
0.5
0.5
0.5
0.5
0.4
T
T
0.5
T
0.5
0.5
T
I
T
0.2
0
1
0
0
0
0
0
0
0
Relative mRNA quantification
Dixdc1
Apc
CylD
Lrp5
Wnt5a
Ndrg1
Cdc73
1.5
2.0
1.5
2.0
1.5
ns
ns
2.5
4
ns
2.0
1.0
1.5
1.0
1.5
T
T
1.0
3
T.
1.5.
1.0
T.
1.0
2
1.0
T
0.5
0.5
0.5
0.5
0.5
0.5
1
T
T
WT
0
0
0
0
0
0
0
Sf1:Cre; Prkar1a Fl/Fl
Supplementary Figure 5. Expression of WNT pathway regulators in response to PKA stimulation. A- Evaluation of the specificity of antibodies detecting NDRG1, CDC73 and WNT4. Top two panels show immunodetection of NDRG1 and CDC73 in wild-type adrenals (a,c) and the corresponding negative controls (b,d, no primary antibody). Although some non-specific vascular staining is present in d, specific nuclear staining for CDC73 is undetectable in the absence of the primary. Lower panel shows immunodetection of WNT4 in a wild-type adrenal (e) and the absence of signal in Sf1:Cre;Wnt4 Fl/Fl adrenal cortex (f). B-C- Axin2 and Wnt4 expression levels are correlated in vivo. Axin2 and Wnt4 expression levels were correlated on the basis of RTqPCR data presented in Fig 3 D & E. D- Short-term ACTH treatment induces Akr1b7 and represses Wnt4 and Axin2 expression in vivo. Wild-type mice were treated with a single injection of ACTH (Synacthene Retard) or vehicle and sacrificed 24h later. Expression of Akr1b7, Axin2 and Wnt4 was then evaluated by RTqPCR. Bars represent the mean of expression in 4 animals per treatment group + SEM. E- H89 treatment induces WNT4 expression and blocks the inhibitory effect of Forskolin. H295R cells were treated with vehicle (DMSO), forskolin (10-5M), H89 (10 µM) or a combination of both for 8h (after 2h preincubation with H89) and expression of WNT4 was analysed by RTqPCR. Bars represent the mean expression in three independent experiments + SEM. F- Wnt4 expression is almost abolished in Sf1:Cre; Wnt 4Fl/FI adrenals. Wnt4 expression was analysed by RTqPCR on mRNAs extracted from wild-type and Sf1:Cre;Wnt4Fl/Fl adrenals. Bars represent the mean expression in 10 adrenals per genotype + SEM. F- Akr1b7 staining area is increased in the adrenal cortex of Sf1:Cre;Wnt4 Fl/Fl mice. AKR1B7 staining in wild-type and Sf1:Cre;Wnt4 Fl/Fl mice was evaluated as in Supplementary Fig. 1C. Statistical analyses were performed by one-way ANOVA followed by Dunnett's multiple comparison test (E) or with Student's t test with Welch correction (D, F-G). * p<0.05; ** p<0.005; *** p<0.0005; NS: Not significant. ZG, zona glomerulosa; ZF, zona fasciculata; M, medulla.
A
With primary Ab.
No primary Ab.
a
b
ZG
Ndrg1
ZG
ZF
ZF
M
M
B
Pearson r = 0.9611 p<0.0001
C
Pearson r = 0.6606 p = 0.03
2.0
2.0
c
ZG
d
Axin2 mRNA
1.5
·
Axin2 mRNA
1.5
Cdc73
ZG
ZF
ZF
1.0
1.0
M
0.5
0.5
M
0
0
WT
Sf1:Cre; Wnt4 Fl/Fl
0
0.5
1.0
1.5
2.0
2.5
0
0.5
1.0
1.5
f
Wnt4 mRNA
Wnt4 mRNA
e
Adipose
Adipose
treated wild-type
mice
Sf1:Cre; Prkaria FI/FI
mice
ZG
Wnt4
ZG
ZF
ZF
M
M
D
Akr1b7
Wnt4
Axin2
E
WNT4
F
Wnt4
G
Akr1b7 surface
Relative mRNA quantification
10
1.5-
1.5-
0.8-
1.0
*
0.5
0
Relative mRNA quantification
-
2.0
Relative mRNA quantification
*
*
*
8
*
0.6
0.8
*
1.0-
1.0
1.5
Ratio to whole
*
6
0.6
0.4-
cortex
4
T
1.0.
0.5
0.5
T
0.4
2
0.2
Ctrl
WT
0.2
WT
0
0
0
ACTH 24h
0
Sf1:Cre; Wnt4 Fl/Fl
0
Sf1:Cre; Wnt4 FI/
+
+
H89
-
+
+
Fsk
-
-
H295R cells
Supplementary Figure 6. Akr1b7:Cre-mediated recombination, DAB2 expression in the adrenal and analysis of Prkaca+/- adrenals. A- Akr1b7:Cre induces LoxP recombination in a subset of cortical cells. Akr1b7:Cre mice were mated with mT/mG reporter mice to generate Sf1:Cre;mTmG offspring. Recombination efficiency was analysed by immunodetection of GFP expression. Sections were also stained with CYP11B2 (ZG) and Hoechst (nuclei). Recombination was induced in a large number of but not all cortical cells, including areas of ZG (inset). B- DAB2 is a bona fide ZG marker. Wild-type adrenals were stained with both CYP11B2 (a) and DAB2 (b) antibodies. Panel c shows the overlap between the two signals in ZG (insets). C- Heterozygosity for Prkaca has no obvious impact on adrenal histology. Adrenals from Prkaca+/+ (WT) and Prkaca+/- were stained with heamtoxylin and eosin. Inset shows high magnification of ZG. ZG, zona glomerulosa; ZF, zona fasciculata; M, medulla.
GFP/Hoechst/Cyp 11b2
B
Akr1b7:Cre; mTmG
Cyp11b2
Dab2
Merge
a
b
c
ZG
ZG
ZG
ZG
ZG
ZG
M
ZF ZG
ZG
ZF
ZF
ZF
M
M
M
C
WT
Prkaca+/-
H&E
Supplementary Figure 7. Activation of WNT pathway is associated with decreased overall survival in ACC patients. WNT activation signature was calculated as the geometric mean of AXIN2, LEF1 and APCDD1 expression levels that were extracted from mRNA sequencing data from TCGA. Patients were separated as two equivalent groups of high (n=39) and low (n=40) WNT activation signature. Left panel shows the Kaplan-Meier estimates of overall survival. In the right panel, patients were ordered by cut-off-centred WNT activation signature. Dark grey boxes indicate specific death.
A
WNT signature: overall survival
TCGA
WNT signature TCGA
median centered mRNA accumulation
1.0
high: 40 cases
4
low: 39 cases
0.8
Frequency
2
0.6
0
0.4
0.2
-2
Dead
logrank test
p-value = 0.0264
HR = 2.37
☐
Alive
0
(95% CI =1.10-5.09)
-4
0
1000
2000
3000
4000
T
I
I
IL
I
I
I
1
Survival (days)
Supplementary Figure 8. ACTH treatment inhibits WNT signalling in ACat mouse adrenals. ACat mice that express a constitutive allele of Ctnnb1 in the adrenal cortex 6 were treated for 5 days with vehicle (Ctrl) or Dexamethasone (Dex) or for 3 days with dexamethasone followed by two days of Synacthene retard (Dex + ACTH). Lef1, Axin2 and Wnt4 expression levels were then analysed by RTqPCR on mRNAs extracted from adrenals. Bars represent mean expression in 4 animals per group + SEM. Statistical analysis was performed with Student's t test. * p<0.05; ** p<0.005; *** p<0.0005.
Lef1
Axin2
Wnt4
2-
*
Relative mRNA quantification
Relative mRNA quantification
Relative mRNA quantification
NS
1.0
1.0
1.
T
0.5
0.5
☒
Ctrl
☒
Dex
T
☐
☐
☐
0-
0
0
☐
Dex + ACTH
Supplementary Figure 9. Non-cropped versions of the western blots shown in Figure 2C, 2D and Supplementary Figure 3.