Untreated
Cisplatin
DAPI
p53
DAPI
p53
S100A4
Overlay
S100A4
Overlay
Supplementary Figure S1. Nuclear co-localization of S100A4 and p53. Fluorescence microscopy images
of S100A4 and p53 in untreated and cisplatin treated A549 cells. Nuclear co-localization of S100A4 and
p53 is exemplified by cells indicated in the image. Nuclei were defined by DAPI staining. Images were
generated using a Zeiss LSM 510 META confocal laser scanning microscope.
Example Image #2
p53+S100A4 Ab (Nutlin)
p53+S100A4 Ab (Ctrl)
S100A4 Ab only (Nutlin)
p53 Ab only (Nutlin)
Example Image #1
Supplementary Figure S2. Example images from in-situ proximity ligation assay of S100A4-p53
interaction in A549 cells +/- Nutlin. As a negative control either S100A4 or p53 antibody was
excluded from the assay. Nuclei were defined by Hoechst staining.
a
b
100%
Dots/cell
>30
11-30
6-10
2-5
0-1
80%
60%
40%
20%
Analyzed cells
Analyzed cells
100%
80%
60%
40%
20%
0%
0%
p53 Ab
(Ms mono)
+
-
+
+
p53 Ab
(Rb mono)
+
-
+
+
S100A4 Ab
(Rb poly)
-
+
+
+
S100A4 Ab
(Ms mono)
-
+
+
+
MG132
+
+
-
+
MG132
+
+
-
+
p53 Ab
(Ms mono)
+
-
+
+
S100A4 Ab
(Rb poly)
-
+
+
+
MG132
+
+
-
+
c
Analyzed cells
100%
80%
60%
40%
20%
0%
Supplementary Figure S3. In-situ proximity ligation assay (PLA) of S100A4-p53 interaction in
cells +/- MG132 treatment. (a) PLA in A549 cells using the same antibody pair as in Figure 2c
(monoclonal mouse anti-p53 antibody (Santa Cruz Biotechnology) and polyclonal rabbit antiS100A4 antibody (Dako)). (b) PLA in A549 cells using another antibody pair (monoclonal rabbit
anti-p53 antibody (Cell Signaling Technology) and monoclonal mouse anti-S100A4 antibody
(DSHB)). (c) PLA in HeLa cells using the same antibody pair as in Figure 2c (monoclonal
mouse anti-p53 antibody (Santa Cruz Biotechnology) and polyclonal rabbit anti-S100A4
antibody (Dako)). In all experiments, as a negative control either S100A4 or p53 antibody was
excluded from the assay. Data represents the number of interaction signals (dots) per cell from at
least 100 cells per condition.
Empty vector
S100A4 shRNA
CHX (min)
0
15
30
60
CHX (min)
120
0
15
30
60
120
p53
S100A4
Actin
Supplementary Figure S4. Immunoblot analysis showing p53 and S100A4 protein levels in A549
empty vector cells and S100A4 shRNA expressing cells treated with cycloheximide (25µg/ml) at
indicated timepoints.
b
a
100%
Dots/cell
>30
11-30
6-10
2-5
0-1
80%
60%
40%
20%
Analyzed cells
Analyzed cells
100%
80%
60%
40%
20%
0%
0%
p53 Ab
(Rb mono)
+
-
+
+
mdm2 Ab
(Ms mono)
+
-
+
+
mdm2 Ab
(Ms mono)
-
+
+
+
S100A4 Ab
(Rb poly)
-
+
+
+
MG132
+
+
-
+
MG132
+
+
-
+
Supplementary Figure S5. In-situ proximity ligation assay (PLA) of p53-mdm2 and S100A4-
mdm2 interaction in A549 cells +/- MG132 treatment. (a) PLA in A549 cells using monoclonal
rabbit anti-p53 antibody (Cell Signaling Technology) and monoclonal mouse anti-mdm2
antibody (Santa Cruz Biotechnology). (b) PLA in A549 cells using monoclonal mouse antimdm2 antibody (Santa Cruz Biotechnology) and polyclonal rabbit anti-S100A4 antibody (Dako).
As a negative control one of the antibodies in each pair was excluded from the assay. Data
represents the number of interaction signals (dots) per cell from at least 100 cells per condition.
800
Empty
vector
700
S100A4
shRNA
Growth (%)
600
500
400
300
200
100
0
0h
24h
48h
Supplementary Figure S6. Relative growth of A549 cells stably expressing S100A4 shRNA
compared to empty vector cells using CellTiter-Blue assay (Promega). The relative growth from
0h timepoint is plotted (n=3 +/- s.d.).
Ctrl
Cisplatin
476
188 (39% of ctrl)
436
61 (14% of ctrl)
Empty
Vector
S100A4
shRNA
Supplementary Figure S7. Colony count related to clonogenic assay shown in Figure 5b. Colonies were
automatically detected and counted by image analysis with the CellProfiler software
(www.cellprofiler.org). Identified colonies are displayed with arbitrary colors to distinguish the separate
colonies.