Supplementary Information
Supplementary Materials and Methods
Soft agar colony formation
Six-well plates were coated with 0.5% (w/v) agarose in growth media and 0.35% (w/v) agarose in media
containing 50 000 cells/well layered on top. Two ml of normal growth media was added per well and replaced
weekly. After three weeks, colonies were counted using a standard bright field microscope. The number of
colonies in four randomised microscopic fields (10x magnification) per assay condition was determined.
Supplementary Figures
r
Draw area of
interest around
core and take
note of area pixel
number (A)
Run positive pixel
count algorithm,
excluding A, and
take note of pixel
number (B)
r
Idealise core as circle with Area A.
Calculate radius r. Calculate area
of band around core with a
thickness of r. This gives the 100%
reference area.
Supplementary Figure 1: Determination of spheroid invasiveness. Images were analysed using Aperio
ImageScope software version 10 (Aperio Technologies, Vista, CA). An area of interest was drawn around the
core spheroid structure and the number of pixels within this area determined. The invaded area was measured
using a positive pixel count algorithm. Next, the core spheroid structure was idealised as a circle with radius r
(calculated from the core spheroid area). A band with radius r was drawn around the idealised core spheroid
structure and the total number of pixels in this band calculated (100% reference area). Finally, the invaded area
pixel count was normalised to the 100% reference area.
Cell Viability (MTT)
WM793
2.5
2
Absorbance
2
1.5
1
KL - DOX
KL + DOX
KM - DOX
KM + DOX
1.5
1
0.5
0.5
p=0.007
5
6
KM +
DOX
***
1205Lu
KL - DOX
-
***
1205Lu
***
KL - DOX
-
% Sub G1
KM + DOX
KM - DOX
7
6
5
4
3
2
1
0
KL - DOX
-
# of Colonies
Relative Signal
KM + DOX
KM - DOX
KL + DOX
KM + DOX
KM - DOX
KL + DOX
KL - DOX
3.5
3
2.5
2
1.5
1
0.5
0
***
KL + DOX
8
7
6
5
4
3
2
1
0
200
180
160
140
120
100
80
60
40
20
0
***
Sub-G1
WM793
-
% Sub-G1
D
KL - DOX
Caspase-Glo 3/7
WM793
KL - DOX
3.5
3
2.5
2
1.5
1
0.5
0
1205Lu
**
-
Relative Signal
C
6
KM + DOX
Colony Formation
WM793
-
# of Colonies
200
180
160
140
120
100
80
60
40
20
0
5
Days
Days
B
4
KM + DOX
4
3
KM - DOX
3
2
KM - DOX
2
1
KM - DOX
1
p=0.022
0
0
KL + DOX
Absorbance
1205Lu
KL + DOX
2.5
KL + DOX
A
Supplementary Figure 2: Functional characterisation of DOX-induced (+MSX2) vs. non-induced control cells
(-MSX2) in two-dimensional cultures of WM793 and 1205Lu cells (sample results including all control cell
lines). (a) Evaluation of cell viability over time using MTT assays. Significance was generated from the average
signal change between consecutive days in three independent experiments. (b) Determination of clonogenic cell
survival. (c) Assessment of caspase 3/7 activation using a bioluminescence-based Caspase-Glo 3/7 assay
system. Luminescence readings were normalised to the total cell number and displayed relative to non-induced
control cells. (d) Sub-G1 cell cycle population as detected via PI staining followed by flow cytometric analysis.
Error bars represent the standard error of the mean within internal replicates. Significance levels were
determined using an unpaired, two-tailed Student’s t-test. KL, cells infected with KRAB and LLCIEP (empty
vector) encoding virus; KM, cells infected with KRAB and MSX2 encoding virus. p-Values refer to KM -/+
DOX results. Significance levels: * p<0.05, ** p<0.01, *** p<0.001.
A
+ MSX2
Count
Count
WM793
- MSX2
Sub G1
1.1%
PI (585 nm)
Sub G1
4.7%
PI (585 nm)
B
15.2%
71.6%
Count
Count
WM793
75.3%
7.2%
PI (675 LP)
18%
6.7%
PI (675 LP)
Count
Count
1205Lu
C
Sub G1
0.4%
PI (585 nm)
Sub G1
5.8%
PI (585 nm)
D
21.1%
12.2%
PI (675 LP)
66.9%
Count
Count
1205Lu
64%
20.1%
10.4%
PI (675 LP)
Supplementary Figure 3: Cell cycle analysis in WM793 and 1205Lu cells in response to MSX2-overexpression. Representative histograms after PI staining and flow analysis of DOX-induced (+ MSX2) vs. noninduced control cells (- MSX2) including the Sub-G1 population of the cell cycle in (a) WM793 cells and (c)
1205Lu cells. Representative histograms after PI staining and flow analysis including viable cells only in (b)
WM793 cells and (d) 1205Lu cells.
A
MSX2
β-actin
B
14
**
% Sub G1
12
*
10
8
6
4
2
0
120
0
0.00010.0005 0.001 0.005 0.01
0.05
100
80
***
60
40
***
***
0.05
20
0.01
# of Colonies
C
0
0.005
0.001
0.0005
0.0001
0
μg/ml Doxycycline
Supplementary Figure 4: Dose-dependent effect of MSX2 expression in WM793 cells. (a) Western blot
analysis of MSX2 protein levels in response to a gradient of DOX (as specified in panel (c)). β-Actin levels
were used to assess total protein loading. Exposure time was adjusted to strongest signal to avoid over-exposure.
(b) Cell cycle analysis by propidium iodide staining and flow cytometry-based evaluation. Values represent the
percentage of cells in Sub-G1, an indicator for cellular apoptosis. (c) Soft agar colony formation assay to test for
clonogenic survival/proliferation of cells in the absence of anchorage. Significance levels: * p<0.05, ** p<0.01,
*** p<0.001.
A
All Subtypes
B
SSM and Nodular
MSX2 positive
(n=40)
MSX2 negative
(n=84)
MSX2 negative
(n=51)
MSX2 positive
(n=80)
p = 0.918
C
p = 0.351
All Subtypes
D
SSM and Nodular
MSX2 positive
(n=80)
MSX2 positive
(n=39)
MSX2 negative
(n=87)
p = 0.990
MSX2 negative
(n=52)
p = 0.700
Supplementary Figure 5: Kaplan-Meier survival analysis of melanoma patients, stratified according to nuclear
MSX2 expression. Recurrence-free survival of patients with (a) all melanoma subtypes and (b) SSM or nodular
melanomas only. Overall survival of patients with (c) all melanoma subtypes and (d) SSM or nodular melanoma
only.
Supplementary Table
Supplementary Table 1: Cox regression analysis of nuclear MSX2 expression and recurrence-free survival or
overall survival (entire cohort).
Prognostic Factor
Recurrence Free Survival
Nuclear MSX2 (pos. vs. neg., ref)
T-Stage (T2-T4 vs. T1, ref)
Ulceration (pos. vs. neg., ref)
Overall Survival
Nuclear MSX2 (pos. vs neg., ref)
T-Stage (T2-T4 vs. T1, ref)
Ulceration (pos. vs. neg., ref)
1
Multivariate1
(95% CI)
p-value
HR
Univariate
(95% CI)
p-value
HR
0.973
7.466
3.267
0.570-1.660
2.689-20.739
1.846-5.781
0.919
<0.001
<0.001
0.807
6.868
2.767
0.453-1.425
2.110-22.362
1.532-4.998
0.460
0.001
0.001
1.003
9.696
3.528
0.587-1.714
3.021-31.117
2.021-6.160
0.990
<0.001
<0.001
0.912
10.350
2.920
0.525-1.587
2.486-43.027
1.627-5.242
0.746
0.001
<0.001
Adjusted for all other variables in the subsection; ref, referent group; HR, hazard ratio; CI, confidence interval