Wnt4
Wnt5A
Wnt3A
Wnt5A
Activated Wnt Signaling Pathway
Inhibited Wnt Signaling Pathway
Wnt Calcium Signaling Pathway
Wnt PCP Signaling Pathway
Wnt
Norrin
DKK1
Wnt5a
Wnt5a
LRP
Frizzled
LRP
Frizzled
Ror
FZD
Ror
FZD
M
JUL
VANGL
Dishevelled
Disheveled
Dishevelled
CKkı
GSK-30
B-Catenin
P
PLC
GSK-30
CKJa
Axin
RAC/ RhoA
Axin
APC
3-Catenin
APC
0
PIP2
IP,
B-Catenin
Ubiquitin- mediated proteolysis
Cytoskeleton remodelling
DAG
Ca2
JNK
B-Catenin
Cell Polarity
TCF/LEF
Gene transcription
TCF/LEF
No gene transcription
5000000
5000000
PKC
CamKII
Calcineurin
Cell Motility
AP-1
NFAT
Gene transcription
0-JUN
Gene transcription
100000
Supplementary Fig. 1: Cartoon depicting activated/inhibited canonical Wnt signaling and non- canonical pathways Wnt-Calcium and Wnt-PCP. Created with Biorender.
WNT3
NFATC2
60-
8-
log2 (count+1)
TVBF-7
log2 (count+1)
TVBF-7
NCI-H295R
40-
MUC-1
6-
NCI-H295R
MUC-1
etr control
20-
JO (+)-JQ1
4-
ctr control
M
THE1 TH21
JO [+)-JQ1
Mitotane
TM THZ1 + Mitotane
2-
1 THZ1
M Mitotane
F
Forskolin
TM THZ1 + Mitotane
Forskolin
0
T
FM Forskolin + Mitotane
FM Forskolin + Mitotane
F
ctr JQ T
M
TM F FM FT
0
ctr
JQ
T
M
TM
FM F FT
ctr JQ
r
M TM
F 1
FM FT
FT Forskolin + THZ1
ctr JQ
T
M
TM F FM FT
ctr JQ
T
M TM F FM FT
ctr JQ
T
M TM F FM FT
FT Forskolin + THZ1
WNT4
NFATC3
200-
20-
log2 (count+1)
TVBF-7
log2 (count+1)
TVBF-7
150
NCI-H295R
15-
NCI-H295R
100
MUC-1
MUC-1
ctr control
10-
ctr control
50-
Je
(+)-J01
JO (+)-J01
P
MI41
₸
1
n
Mitotane
5-
TM THZ1 + Mitotane
M
THZ1
Mitotane
Y
-
S
TM THZ1 + Mitotane
- L
F
Forskolin
Forskolin
FM Forskolin + Mitotane
FT Forskolin + THZ1
0
3
FM Forskolin + Mitotane
ctr
JQ
T M
TM
F E FM FT
ctr JQ
7
M TM
FM FT
ctr JQ
M
TM 1 F FM FT
ctr
JQ
S
M
TM FM F FT
ctr
JQ
T
M TM
F
1 FM FT
ctr
JQ
1
M
TM
1 F FM FT
FT Forskolin + THZ1
WNT5A
NFATC4
100-
80.
log2 (count+1)
TVBF-7
log2 (count+1)
TVBF-7
80
NCI-H295R
60
NCI-H295R
60-
MUC-1
MUC-1
ctr control
40
etr control
40-
JO [+]-JQ1
₸ THZ1
JQ (+)-JQ1
20-
M
TM THZ1 + Mitotane
20-
₸ THZ1
Mitotane
TM THZ1 + Mitotane
M
Mitotane
F
Forskolin
M
Forskolin
0
FM Forskolin + Mitotane
0
FM Forskolin + Mitotane
ctr
JQ
T M TM
FM FT
ctr JQ
1 T
M TM F FM FT
ctr
IQ
T
M
TM
F FM F FT
FT Forskolin + THZ1
ctr JQ
T
M TM
FM FT
ctr
JQ
T
M
TM
F FM FT
ctr
IQ
T M TM F FM FT
FT Forskolin + THZ1
¥ 08
ROR1
MYCN
80
log2 (count+1)
TVBF-7
2.5-
NCI-H295R
log2 (count+1)
2.0-
T
TVBF-7
NCI-H295R
30-
MUC-1
1.5-
MUC-1
etr control
etr control
20
JQ (+)-Q1
THE1
1.0-
(+)JQ1
Mitotane
M
TH21
10
TM M THZ1 + Mitotane
0.5-
Mitotane
TM THZ1 . Mitotane
FM Forskolin + Mitotane
F
Forskolin
0
0.0
FM Forskolin + Mitotane
F
ctr
JQ
T
M
TM
F FM MFT
ctr
JQ
T
M
TM
- FM FT
ctr JQ
T
M TM F FM FT
FT Forskolin + THZ1
ctr
r JQ T
M TM
FM FT
H
ctr JQ
T
M TM
F FM FT
ctr JQ
I
M TM F FM FT
FT Forskolin + THZ1
FZD1
LGR5
150
TVBF-7
250
log2 (count+1)
NCI-H295R
log2 (count+1)
TVBF-7
200.
100
NCI-H295R
MUC-1
150
MUC-1
etr
control
control
50-
()-JQ1
M
THZ1
100
C (+)JQ1
Mitotane
T
TM M THZ1 + Mitotane
50-
TA THZ1 + Mitotane
M
TH21
Mitotane
F
Forskolin
0
Z M TM
·
V
S
FM M Forskolin + Mitotane
R Forskolin
FM Forskolin + Mitotane
1
0
ctr J
JQ
1 FM
FT
ctr
JQ
T
M
TM
3
1
FM
FT
ctr JQ
L
M
TM
F
1 FM FT
FT Forskolin + THZ1
1 J tr JQ
1
M
TM F
FM
IFT
Y
ctr
JQ
T
M TM
E FM F
ctr JQ 1
M
TM
FM F
T
FT Forskolin + THZ1
VANGL1
DVL1
200.
80
log2 (count+1)
TVBF-7
log2 (count+1)
TVBF-7
150-
NCI-H295R
60
NCI-H295R
MUC-1
MUC-1
100-
otr control
40
ctr
control
[*]-JQ1
C (*)01
50-
T
TH21
Mitotane
T THZ1
TN THZ1 + Mitotane
M
20-
M Mitotane
TN THZ1 + Mitotane
1
FM Forskolin + Mitotane
F
3
FM Forskolin + Mitotane
F
0
ctr
JQ
1
M
TM
- FM
FT
ctr
JQ
T
M
TM
FT
ctr JQ
M
TM
1 FM FT
FT Forskolin + THZ1
0
ctr
JQ
T
M
TM F
FM
FT
ctr JQ
1
M
TM E FMFT
L
ctr JQ
T
M TM
F FM FT
FT Forskolin + THZ1
DVL2
JUN
150
1500-
log2 (count+1)
TVBF-7
log2 (count+1)
TVBF-7
NCI-H295R
NCI-H295R
100-
MUC-1
1000-
MUC-1
ctr control
50-
JO (IJ01
ctr control
JA
MASO TH21
M
TH21
500-
Mitotane
TM THZ1 + Mitotane
Mitotane
F
Forskolin
TM THZ1 + Mitotane
FM Forskolin + Mitotane
F
Forskolin
0
ctr JQ
S
FM Forskolin + Mitotane
0
M
TM
1 FM FT
ctr
JQ
T
M
TM
F
F FMFT
ctr
JQ
T
M
TM
FM FT
FT Forskolin + THZ1
ctr
JQ
f
M
TM
FM FT
ctr
JQ
M TM
F FM FT
ctr JQ
T
M TM F FM FT
FT Forskolin + THZ1
DVL3
MYCL
600
10-
log2 (count+1)
TVBF-7
log2 (count+1)
TVBF-7
NCI-H295R
8-
NCI-H295R
400
MUC-1
6-
MUC-1
etr control
Ger tr control
200-
JO (+)-JQ1
1
THZ1
4-
JQ (*)-JQ1
M
Mitotane
I
TH21
TM THZ1 + Mitotane
2-
Mitotane
FM Forskolin + Mitotane
F
Forskolin
TM THZ1 + Mitotane
F
Forskolin
0
0
FM Forskolin + Mitotane
ctr JQ
M TM
FM FT
ctr JQ
1
M
TM
1 F FM FT
ctr
JQ
T
M
TM
F FM F
T
“T Forskolin + THZ1
ctr
JQ 1
T
M TM
5
FM FT
ctr
JQ
M
TM
F
I. FMFT
ctr JQ
T
M TM
FM FT
FT Forskolin + THZ1
LEF1
LGR4
400
A
log2 (count+1)
TVBF-7
500
300
200
NCI-H295R
log2 (count+1)
TVBF-7
400
NCI-H295R
100
MUC-1
300-
MUC-1
60
tr control
JO (*)-01
etr control JQ (*)-JQ1
40-
T 1
200
M
TH21
Mitotane
₸ TH21
M
20
TM THZ1 + Mitotane
Mitotane
FM Forskolin + Mitotane
F
Forskolin
100
TM THZ1 + Mitotane
0
Forskolin
L
0
R
FM Forskolin + Mitotane
ctr JQ
M TM
1 FM FT
ctr
JQ
T
M
TM
F FM FT A
ctr
JQ
C
T
M TM
₾
FM FT
FT Forskolin + THZ1
ctr
JQ
A
TM
F FM FT
ctr
JQ
1
M
TM
IFT
FM F
ctr
JQ
1
M TM
F FM FT 1
FT Forskolin + THZ1
MYC
600
log2 (count+1)
TVBF-7
400-
NCI-H295R
200- -
MUC-1
80
etr control
S
JQ (+)-JQ1
T THZ1
40
Mitotane
TM THZ1 + Mitotane
M
20
Forskolin
0
FM Forskolin + Mitotane
ct
10
A
-
₹
N
C
7
1
ctr
0
A
N
2
A
FT
ctr
10
-
2
1 FM FT
FT Forskolin + THZ1
AN
Supplementary Fig. 2: High magnification of the gene expression data presented in Fig. 1 of selected signaling components of Wnt-B-catenin, Wnt-Ca2+, Wnt-PCP under different conditions and for th three different models TVBF-7, NCI-H295R and MUC-1. From left to right: untreated controls (C), JQ1 (J), THZ-1 (T), mitotane (M), THZ-mitotane (TM), forskolin (F), forskolin-mitotane (FM), forskolin-THZ1 (FT), (n=3).