A

ATM

-actin

scRNA siATM

B

Supplementary Figure S1. (A) H1299 cells harboring pGC were transfected with I-SceI-expressing

vector. Percentage of GFP+ cells was assessed after inhibition of ATM (10µM KU55933), MRE11 (25µM

Mirin) or both 48h post-transfection as an indication for HR efficiency. (B) inhibition of MRE11 does not

affect ATM signaling. A549 cells were treated with 25µM mirin for 2h prior to irradiation with 3Gy and after

30 min phosphorylation of both ATM at S1981 and CHK1 at Thr 68 were investigated. (C) Western blot

analysis showing efficient siRNA–mediated knockdown of both ATM (left panel) and MRE11 (right panel).

0.00

0.05

0.10

0.15

ATMiMRE11i

H1299-pGCInhibition

HR

effici

ency

(% G

FP+ c

ells

)

CMRE11

-actin

scRNA siMRE11

0

5

10

15

RAD

51/

H2A

X fo

ci p

er c

ell

IRsiATMsiMRE11siATM / siMRE11

3h 24h

Supplementary Figure S2. After irradiation with 2Gy, RAD51 colocalized with γH2AX foci were

monitored in A549 cells after ATM knockdown (siATM), MRE11 (siMRE11) or both

(siATM/siMRE11). Knockdown of ATM or MRE11 should a significant reduction in RAD51 foci at

3h time point after 2Gy. Knockdown of both proteins did not show any synergistic effect on the

number of RAD51 foci at this time point. At the 24h time point, the number of residual RAD51 foci

was significantly higher upon ATM knockdown than after MRE11 knockdown. Simultaneous

depletion of both proteins showed a slight increase in RAD51 compared to ATM depeltion alone.

In all cases, the number of foci measured in non-irradiated cells was subtracted from that

observed in irradiated cells. Error bars represent the SEM of three independent experiments.

0 1 2 3 4

0.1

1

IRATMi 2h post-IRATMi 4h post-IRMRE11i 2h post-IRMRE11i 4h post-IR

ATMi/MRE11i 4h post-IRATMi/MRE11i 2h post-IR

X-ray dose (Gy)

Surv

ival

frac

tion

Supplementary Figure S3. Radiosensitivity of exponentially growing A549 cells was

measured by colony forming assay after inhibition of ATM (10µM KU55933) and/or MRE11

(25µM Mirin) either 2h (2h-post) or 4h (4-post) after the indicated X-ray doses. Error bars

represent the SEM of three independent experiments.

A B0Gy

2Gy2h 4h 6h 8h

Cenp

FRA

D51

Cenp

FRA

D51

Cenp

FRA

D51

cont

rol

ATM

i pre

ATM

i pos

t

2 4 6 80

10

20

30

S/G2 cells (CenpF+, APH)

IRATMi pre-IRATMi post-IR

Time interval after 2Gy, h

RAD

51 fo

ci p

er c

ell,

rela

tive

Supplementary Figure S4. (A) Representative IF photos for RAD51 foci (Red) in CenpF+ S/G2 (green) A549

cells at the indicated time points after ATM inhibition either 1h prior to (-pre) or 2h after (-post) 2Gy. 5µM

Aphidicoline (APH) was added to block the transition of S-phase cells into G2. (B) Quantification of RAD51

foci in at least 50 CenpF+ cells for each indicated time point. In all cases the number of foci measured in non-

irradiated cells was subtracted from the number of IRIF observed in irradiated cells. Error bars represent the

SEM of three independent experiments.

A

B

Supplementary Figure S5. (A) Identification of cells in different cell‐cycle phases in A549 cells.

Cells were pulsed labeled with EdU and co-stained with CenpF. Using these two markers the

different cell cycle phases are distinguished (G1-phase cells: CenpF-negative, S-phase cells:

CenpF-positive and EdU-positive, G2-phase cells: CenpF-positive and EdU-negative). (B)

Examples of discrete RAD51 foci in CenpF+ and EdU- G2 cells (left panel) or CenpF+ and EdU+

S-phase cells (right panel) at the indicated time points after 2 Gy of X-ray.

0

10

20

30IRATMi pre-IRATMi post-IR

4h

S/G2 cells (CenpF+)

siKAP1 + + +

8h

+ + +

RAD

51 fo

ci p

er c

ell,

rela

tive

Supplementary Figure S6. (A) Western blot showing an efficient KAP1 depletion using

siRNA (+siKAP1) in A549 cells. (B) ATM was then inhibited in A549 cells either 1h prior to

(ATMi-pre) or 2h after (ATMi-post) 2Gy and RAD51 foci (relative to mock irradiated cells)

were monitored in CenpF+ S/G2 cells at the indicated time points. Non-specific siRNA was

used as a control (- siKAP1). In all cases the number of foci measured in non-irradiated cells

was subtracted from the number of IRIF observed in irradiated cells. Error bars represent the

SEM of three independent experiments.

A

B

KAP1

-actin

scRNA siKAP1

0

5

10

15

20

25

SCE

per

met

apha

se

***

**

ATMi + pre post

2GyUT

Supplementary Figure S7. Detection of sister chromatid exchanges after inhibition of ATM

either prior to or after 2Gy. A549 cells were treated with or without ATMi prior to or after 2Gy

then the cells were incubated overnight with colcemid (added at 5 h after irradiation) to collect

cells in mitosis. (A) Representative example of a metaphase spread shows multiple SCE

(indicated by arrows). (B) quantification of SCE per metaphase spread in un-irradiated (UT) or

irradiated (2Gy) cells. At least 40 metaphases per data point were analyzed.

A B

Supplementary Figure S8. Detection of apoptosis in via caspase activity. ATM was inhibited in

A549 cells either prior to (pr-IR) or 2h after (post-IR) X-irradiation with 2 Gy. Caspase activity (as

indicator for apoptosis) was then analyzed at 24h time point. Values obtained for non-irradiated

cells were subtracted. A549 or Jurkat cells were irradiated with 2 Gy and incubated with 1 μM

staurosporine for 24 h were used as a positive control.

0

5

10

15

20

40

60

80

100

casp

ase

activ

ity (%

of c

ells)

ATMiMRE11i

--

+-

-

+

IR (2Gy) - - -

++

-

--

+

+-

+

-+

+

++

+

+-

+

-+

+

++

+

Pre -IR Post -IR Juka

rt +

Sta

u.

+ St

au.

A549 cells