Supplementary Figure 1. DAXXS564A does not change Mdm2/p53 stability or p53 mediated gene ‑expression. (A) U2OS cells stably transduced with pCDH-DAXXWT or pCDH-DAXXS564A vectors were treated with 40 μM VP16 for 0, 2, 4 or 6 hours and RNA expression of indicated p53-dependent genes was analyzed by qRT PCR.‑ Expression values were normalized to the average of two reference genes (β-actin and GAPDH). (B) U2OS cells were transfected with empty FLAG-CMV (EV), FLAG tagged wild-type DAXX (WT), single S564A, S564E or quadruple mutant as indicated and treated with 8 nM NCS for 8 hours. Cells were then collected after the treatment with 40 μl/ml CHX at the indicated times and subjected to western blot analysis using labeled antibodies.

FLAG M2

Mdm2

p53

HAUSP

0 10 8030 0 10 8030 0 10 8030 10 8030 0 10 8030 0 10 300 80

EV WT S564A S564E

S564A S707A S712A T726A

-

CHX (min)

Noxa Mdm2 Wip1 Sesn2 p21 Puma0

1

2

3

4

5

6

7

8

9WT_0 hWT_2 hWT_4 hWT_6 hS564A_0 hS564A_2 hS564A_4 hS564A_6 h

B

A

B

A

Supplementary figure 2. Analysis of U2OS clones with potential DAXX depletion. Western blot analysis of DAXX expression was done on 165 U2OS clones obtained after the transfection with TALEN constructs and subsequent sub-cloning to get one founder cell in each well of 96 wells ‑plate. Promising clones with low or undetectable DAXX were chosen and further used together with several clones possessing normal DAXX level that would serve as a control. In further analysis, the DAXX expression was detected by both (A) qRT-PCR and (B) western blot with the use of various DAXX antibodies. Only the clones with low DAXX mRNA level and in which DAXX could not be detected with all used antibodies were considered as the clones with DAXX knock-out (namely 17-7, 17-18 and 17-42).

130

130

DAXX (02+03)

Ponceau

DAXX (M-112)

0-4 0-180-

3 0-7 0-8 0-110-

160-17

0-26

0-43

17-3

17-5

17-6

17-7

17-18 1

7-24

17-26

17-36

17-37

17-40

17-42

17-44

17-46

17-48

17-54

17-61

17-64

17-66

P-13

P-14

U2OS clones

130DAXX (25C12)

0-40-18 0-3 0-7 0-8

0-110-16

0-170-26

0-4317-3

17-517-6

17-717-18

17-2417-26

17-3617-37

17-4017-42

17-4417-46

17-4817-54

17-6117-64

17-66P-13

P-14Ctrl

.Ctrl

.0

1

2

3

4

5

6

Rela

tive

DAXX

mRN

A le

vel

IP: FLAG M2

Input

P-(S/T) ATM/ATR substrate

P-Chk2 (T68)

-tubulin

NCS

+ ATMiWT WT S707A S712A T726AWT

DAXX

S564A

S564A S707A S712A T726A

P-(S/T) ATM/ATR substrate

P-Chk2 (T68)

U2OSVP16 UV

DAXX

-

P-p53 (S15)

ATM

shATM U2OSVP16 UV-

H2AX

P-Chk1 (S317)

Chk2

IP: FLAG M2

Input

B

Supplementary figure 3. Phosphorylation of DAXX at S564 is ATM-dependent. (A) HEK 293T cells were transfected with expression constructs encoding FLAG-tagged wild-type DAXX (WT), DAXXS564A, DAXXS707A, DAXXS712A or DAXXT726A and treated with 8 nM NCS for 1 hour. Cells transfected with FLAG-DAXXWT were also pretreated with 10 μM ATM inhibitor KU-55933 (ATMi) 30 min before DNA damage. Cells were lysed and FLAG-tagged proteins immunoprecipitated with anti-FLAG M2 beads and analyzed by western blotting with antibodies against phosphorylated (S/T) ATM/ATR substrate, DAXX, phosho-Chk2 (T68 )or α-tubulin. (B) U2OS and U2OS shATM cells transfected with FLAG-DAXXWT were treated with 10 μM VP16 or exposed to UV (20 J/m2) and collected 1 hour later. Cells were lysed, immunoprecipitated using anti-FLAG M2 beads and analyzed by western blot using indicated antibodies.

A

Wip1

U2O

S

MCF

7

BJ

100 kDa

70 kDa

-tubulin

BA

P-p53 (S15)

0 m

in

30 m

in

1 h

3 h

5 h

11 h

0 m

in

30 m

in

1 h

3 h

5 h

8 h

8 h

11 h

P-DAXX (S564)

-tubulin

Wip1

si Luc si Wip1

H2AX

Supplementary figure 4. The impact of Wip1 on phosphorylation of its substrates in BJ fibroblasts. (A) BJ cells were transfected with control (si Luc) or siRNA targeting Wip1 and 3 days later exposed to 5 Gy of IR. Cells were lysed at the indicated time points after DNA damage and analyzed by western blotting with the indicated antibodies. α tubulin was used as a loading control. (B) To ‑demonstrate the size and expression level of endogenous Wip1 protein, non-treated, normally growing U2OS, MCF7 and BJ cells were lysed and subjected to western blot analysis using antibodies against Wip1 and α tubulin. Note the aberrantly migrating abundant mutant Wip1 ‑(around 70 kDa), and overexpressed wild-type Wip1 protein (the top band in Wip1 blots) in U2OS and MCF7 cells, respectively, compared to low levels of wild-type Wip1 seen in human BJ fibroblasts (see ref. 58 for further discussion on aberrant Wip1 in cancer cell lines).