Supplemental Material
Supplemental Figure legends
A
EU
PC4
RPA
DAPI
EU
RNA POL II
S2
RPA
DAPI
before
0s
10 s
control
HU 2 mM
HU 10 mM
70
60
50
40
HU + DRB
HU
80
30
20
HU
% cells > 9 GFP-PC4 foci
C
B
10
HU + DRB
0
D
D
shRNA 1
rabbit-a-PC4
DAPI
GFP
shRNA 2
rabbit-a-PC4
DAPI
GFP
shRNA 3
rabbit-a-PC4
DAPI
cherry
rabbit-a-PC4
DAPI
cherry
rabbit-a-PC4
DAPI
cherry
rabbit-a-PC4
DAPI
+ Dox 120 h + Dox 96 h + Dox 72 h
control
+ Dox 120 h + Dox 96 h + Dox 72 h
control
GFP
shRNA 1
Dox
-
48 h
72 h
shRNA 2
96 h
-
48 h
72 h
shRNA 3
96 h
-
48 h
72 h
96 h
PC4
tubulin
Figure S1. Effect of various inhibitors and R-loops on PC4 recruitment
and generation of inducible PC4 shRNA cell lines. (A) Resection,
checkpoint or transcription inhibitors were added 1 h before addition of HU (2
or 10 mM for 24h). HU was washed away after 24 h and damaged cells were
fixed after the indicated time periods and analysed for GFP-PC4 foci
formation. Images were taken at a high throughput microscope and
percentage of cells with more than 9 foci/cell quantified using the InCell
Investigator software. (B) U2OS cells were treated with 50 µM DRB and 2 mM
HU for 24 h. 1 h before fixation 0.2 mM EU was added to the cells. Fixed cells
were stained for EU, PC4, RPA and RNA Pol II S2 (marker for elongating
RNA Polymerase II). Representative confocal images are shown. (C)
Overexpression of GFP-RNAse H1 does not affect recruitment of RFP-PC4 to
laser tracks. U2OS cells expressing either RFP-PC4 alone or together with
GFP-RNAse H1 were microirradiated and accumulation of RFP-PC4 at DNA
damage sites followed over time. (D) Analysis of Doxycycline inducible PC4
knockdown cell lines. U2OS cell lines stably transfected with three different
PC4 shRNA constructs either co-expressing GFP or Cherry were incubated in
medium containing 1 µg/ml Doxycyline for the indicated time periods, fixed
and stained for expression of PC4. All three GFP (top panel) and Cherry
(bottom panel) expressing stable U2OS cell lines show knockdown of PC4
after induction with Doxycycline. In addition, GFP-expressing Doxycycline
inducible PC4 knockdown U2OS cells were incubated in medium containing 1
µg/ml Doxycyline for the indicated time periods, harvested and analysed for
expression of PC4 by Western Blot. All three GFP cell lines exhibit
knockdown of PC4 after induction with Doxycycline.
A
BrdU
PC4
B
DAPI
D
F
*
1.3
1.1
0.9
0.7
- HU
+ HU
+ Dox
- HU
Relative PC4 intensity
1
+ HU
***
***
0.8
0.6
0.4
0.2
+
GFP-PC4
No.2
No.3
-
+
-
+ HU
6
- Dox
+ Dox
5
4
3
2
1
0
- HU
+ HU
1.5
1.3
1.1
0.9
0.7
0.5
- HU
-
1
- HU
0
1 µg/ml Dox
2
E
Relative RAD51 intensity
C
PC4
shRNA1
3
0
0.5
G
*
4
Relative gH2AX intensity
Relative RPA intensity
+ HU
- Dox
- HU
Relative BrdU intensity
1.5
GFP-PC4
1-61
+
-
- HU
+ HU
+
GFP-PC4
62-127
-
+
+ HU
GFP-PC4
W89A
-
+
GFP-tagged
PC4
Endogenous
PC4
TUBULIN
H
I
shRNA1 - Dox
shRNA1 + Dox
PC4 shRNA 1
350000
PC4 shRNA 1 +Dox
300000
GFP-PC4
Cell number
250000
GFP-PC4 +Dox
200000
GFP-PC4 1-61
150000
GFP-PC4 1-61 +Dox
100000
GFP-PC4 62-127
GFP-PC4
GFP-PC4 1-61
GFP-PC4 62-127 +Dox
50000
GFP-PC4 W89A
0
day 1 day 3 day 5 day 8
GFP-PC4 62-127
GFP-PC4 W89A
GFP-PC4 W89A +Dox
Figure S2. Induction of ssDNA in PC4 knockdown cells and
characterisation of PC4 rescue cell lines. (A) Representative images of
BrdU and PC4 in control and PC4 depleted cells. PC4 shRNA expression was
induced by incubating cells in medium containing 1 µg/ml Doxycycline for 72h.
BrdU was added 24h before fixation or HU treatment and detected omitting
the denaturation step to identify ssDNA. Relative intensities of (B) BrdU, (C)
PC4, (D) RPA, (E) RAD51 and (F) H2AX. Data from two independent
experiments with at least two replicates are shown. (G) Western Blot of cell
lines expressing different GFP-tagged PC4 versions. Original Doxycyclineinducible PC4 shRNA U2OS cell line (shRNA1) and derived clones stably
expressing shRNA resistant GFP-tagged wildtype PC4 (GFP-PC4 clone 2 and
3), truncated versions of PC4 (aa 1-61 or aa 62-127) or a ssDNA mutant
(GFP-PC4 W89A) were incubated in normal medium or in medium
supplemented with 1 µg/ml Doxycyline (Dox) for 72 h before collection for
Western Blot analysis. An antibody raised against the N-terminus of PC4 was
used for detection, explaining the weak band observed for GFP-PC4 62-127,
lacking this domain. (H) Proliferation assay of PC4 knockdown and rescue cell
lines. (I) FACS profile of wildtype, PC4 knockdown and rescue cell lines.
-Dox
+Dox
-Dox
+Dox
% cells
9 53BP1
foci foci
% >cells
> 9 53BP1
% cells
9 gH2AX
foci foci
% >cells
> 9 gH2AX
100
A
80
100
60
80
40
60
***
20
40
***
200
**
**
*
***
***
***
*
***
***
***
***
B
***
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
35
90
80
70
90
60
80
50
70
40
60
30
50
20
40
10
30
200
10
0
***
*** ***
***
*** ***
***
***
% cells
9 RAD51
foci foci
% >cells
> 9 RAD51
30
35
25
30
20
25
15
20
10
15
5
10
0
5
40
**
0
4
8
24
80
70
60
50
40
30
20
10
0
40
***
20
0
c
48
0
0
4
8
24
48
*
0
4
8
80
70
60
50
40
30
20
10
0
24
c
% cells > 9 Rad51 Foci
**
5
0
4
8
30
20
10
0
c
24
time (h) after release
8h
24 h
*
0
4
8
24
48
80
70
60
50
40
30
20
10
0
60
40
*
0
0
4
8
24
30
15
10
5
0
4
8
24
**
0
4
8
24
48
50
40
30
20
10
*
0
35
30
25
20
15
10
5
0
0
4
8
24
48
*
c
48
0
4
8
24
48
time (h) after release
time (h) after release
E
F
6
Caspase 3 rel Int
48
time (h) after release
*
0
24
*
c
20
c
48 h
8
*
c
48
25
48
4
time (h) after release
80
20
0
*
time (h) after release
10
0
40
time (h) after release
100
c
20
c
48
*
48
25
control
24
**
time (h) after release
15
8
*
50
time (h) after release
% cells > 9 RPA Foci
% cells > 9 RPA Foci
70
60
50
40
30
20
10
0
4
60
time (h) after release
*
c
*
% cells > 9 γH2AX Foci
0
H2O2 1 mM 10 min
% cells > 9 RPA Foci
20
c
% cells > 9 Rad51 Foci
4
**
**
60
time (h) after release
pDNA-PK
- Dox
0
14%
12%
10%
8%
6%
4%
2%
0%
5
4
3
2
1
0
c
0
4
8
24
time (h) after HU release
48
**
8
24
time (h) after HU release
**
**
% cells > 9 γH2AX Foci
γH2AX
% cells > 9 γH2AX Foci
c
80
time (h) after release
+ Dox
***
***
**
% cells > 9 53BP1 Foci
60
c
RPA
***
CPT 50 nM 24 h
80
% cells > 9 53BP1 Foci
53BP1
% cells > 9 53BP1 Foci
VP-16 1 µM 24 h
RAD51
***
***
*
***
***
0
C
D
***
***
***
% cells > 9 Rad51 Foci
% cells
5 RPA
% >cells
> 5foci
RPA foci
PC4 rel int
0
90
80
70
90
60
80
50
70
40
60
30
50
20
40
10
30
200
10
0
- Dox
+ Dox
new
origins
stalled
forks
control
new
origins
stalled
forks
2 mM HU 2 h
48
Figure S3. PC4 knockdown results in impaired repair, induction of
apoptosis and activation of dormant origin firing upon HU treatment. (A)
Foci formation of different DNA damage markers in wild-type and PC4
knockdown cells treated with different concentrations of HU, VP-16 and CPT
for 2 or 24 h. U2OS cells containing a stably integrated inducible PC4 shRNA
construct were seeded in 96 well plates and treated with the indicated drugs
and doses after 3 days incubation in medium with or without 1 µg/ml Dox.
Mean values of at least 9 independent experiments are shown for γH2AX,
53BP1, RPA and RAD51. Knockdown of PC4 in otherwise untreated cells
was sufficient to induce DNA damage or prevent repair of pre-existing lesions,
revealed by spontaneous phosphorylation of H2AX as well as 53BP1 and
RPA foci formation. (B) Relative PC4 intensity in wildtype and PC4
knockdown cells shows stable knockdown of PC4 over 48 h in Dox treated
cells. (C) Defective repair in PC4 knockdown cells treated with VP-16, CPT or
H2O2. Wildtype (- Dox) and PC4 knockdown (+ Dox) cells were treated with
VP-16, CPT or H2O2 and DNA repair kinetics were analysed after release
from the drugs by fixing cells at the indicated time points and staining for
. Images were taken with a high throughput
microscope and percentage of cells with more than 9 repair foci/cell was
determined using InCell Analyzer software. Mean values of more than 3
independent experiments are shown. Note the general trend of elevated
γH2AX, 53BP1, RPA and RAD51 foci formation and delayed repair in PC4depleted cells treated with VP-16, CPT or H2O2. (D) Representative images of
pDNA-PK IF staining in wildtype and PC4 knockdown cells treated with 2 mM
HU for 24 h reveals increased checkpoint activation in PC4 depleted cells. (E)
PC4 knockdown results in increased apoptosis. Cells were depleted for
endogenous PC4 and DNA damage was induced by adding HU for 24 h,
followed by fixation and staining for Cleaved Caspase 3 and DAPI. (F)
Increased origin firing in PC4 knockdown cells upon induction of replication
stress. Wildtype and PC4 depleted cells were treated with 2 mM HU for 24 h
and the number of new origins and stalled forks was determined using the
fibre assay. Scale bar, 10 µm. Error bars represent SEM. Statistical
significance was determined with Student’s t-Test. *p<0.05, **p<0.01,
***p<0.001. Black diamonds = +Dox, White squares = -Dox.
A
Relative Survival
1.2
- Dox
1
0.8
+ Dox
0.6
GFP-PC4 62-127
0.4
GFP-PC4 W89A
0.2
0
control
1 Gy
3 Gy
6 Gy
9 Gy
B
% cells > 9 53BP1 Foci
% cells > 9 γH2AX foci
C
100
80
**
60
***
40
20
**
0
c
0.5
4
8
*
**
10
5
0
c
F
0.5
4
8
*
*
0
0.5
4
8
24
30
*
25
20
15
**
10
*
5
0
c
24
8
0.5
4
8
24
time (h) after Irr
G
time (h) after Irr
50
% cells > 9
CHK2 T68 foci
% cells > 9
CHK1 S345 Foci
20
time (h) after Irr
% cells > RAD51 foci
cells > 9 RPA foci
*
**
**
40
E
20
15
*
60
c
30
25
80
24
time (h) after Irr
D
100
6
*
4
*
2
40
*
30
20
10
0
0
c
0.5
4
8
time (h) after Irr
24
c
0.5
4
8
24
time (h) after Irr
Figure S4. PC4 depletion leads to reduced survival and repair after γIrradiation. (A) Cellular survival of wildtype (- Dox), PC4 knockdown (+ Dox)
and rescue cells expressing either GFP-tagged N-terminal PC4 (GFP-PC4
62-127) or a ssDNA binding mutant (GFP-PC4 W89A) after γ-Irradiation was
determined using the Resazurin assay. Depletion of endogenous PC4 was
induced by incubating cells in medium containing 1 µg/ml Dox for 72 h. (B)
Wildtype (- Dox) and PC4 knockdown (+ Dox) cells were irradiated with 2 Gy
and DNA repair kinetics were analysed by fixing cells at the indicated time
points. Images were taken with a high throughput microscope and percentage
of cells with more than 9 foci/cell of (B) γH2AX, (C) 53BP1, (D) RPA, (E)
RAD51, (F) CHK1 S345 and (G) CHK2 T68 was determined using InCell
Analyser software. Mean values of 3 independent experiments are shown.
Error bars represent SEM. Statistical significance was determined with
Student’s t-Test. *p<0.05, **p<0.01, ***p<0.001. Black diamonds = + Dox,
White squares = - Dox.