Disruption of the MDM2-p53 interaction strongly potentiates p53-dependent apoptosis in

cisplatin resistant human testicular carcinoma cells via the Fas/FasL pathway

Roelof Koster, Hetty Timmer-Bosscha, Rainer Bischoff, Jourik A. Gietema and Steven de Jong

Supplemental Figure 1. Immunofluorescence showing cytoplasmic localised p53, while MDM2

maintains nuclear localised after cisplatin treatment (8 mM) in the cisplatin-resistant TC cell

lines Tera-CP, Scha and 2102EP; a representative example of three independent experiments is

shown. Selected area of the original image, as indicated, x2.5 digitally magnified. Scale bar: 30 mm.

Untreated Cisplatin (8 �M) Untreated Cisplatin (8 �M) Untreated Cisplatin (8 �M)

Tera-CP Scha 2102EP

MDM2 p53

Hoechst

Supplemental Figure 2. (a) No induction of p53 is observed after treatment with RITA. Suppression of p53 with

a specific siRNA does not interfere with the apoptosis induction by RITA as visualised by PARP cleavage; a

representative example of three independent experiments is shown. (b) Immunofluorescence shows that p53

becomes more prominently nuclear localised after Nutlin-3 treatment in the cisplatin-sensitive TC cell line

Tera and the intrinsically cisplatin-resistant cell line 2102EP compared to control; a representative example

of three independent experiments is shown. Selected area of the original image, as indicated, x2.5 digitally magnified. Scale bar: 30 mm.

PARP

p53

+ +- -- Nutlin-3 (4 �M)- - -

+- -- RITA (0.5 �M)-+ + +

Actin

+- -- siRNA p53- +- -

Tera NCCIT

Tera

DMSO Nutlin-3 (4 �M)

2102E

P

Mdm2

p53

a b

Supplemental Figure 3. (a) Successful downregulation of p53 using siRNA targeting p53, and decreased

cleavage of PARP in p53-suppressed Nutlin-3 treated TC cells compared to control; a representative example

of three independent experiments is shown. (b) Unchanged Fas membrane expression of the mutant p53 cell

line NCCIT following Nutlin-3 and/or cisplatin treatment; a representative example of three independent

experiments is shown. (c) Reduced PARP cleavage in Tera-CP and Scha after successful downregulation of

FasL or blocking of FasL, with Nok-1; a representative example of three independent experiments is shown.

(d) Immunofluorescence shows that p21 is mainly cytoplasmic localized after DMSO (control) and Nutlin-3

treatment in the cisplatin-resistant TC cell line 2102EP; a representative example of three independent expe-

riments is shown. Selected area, as indicated, of the original image x4 digitally magnified. Scale bar: 30 mm.

(e) Downregulation of p21 increases the apoptotic response of the intrinsically cisplatin-resistant cells Scha

and 2102EP after Nutlin-3 treatment; a representative example of three independent experiments is shown.

+ + + +- - - - Nutlin-3 (4 �M)

p21

PARP

- + - +- + - + p21 siRNA

Actin

Scha 2102EP

e

- + + + +

Tera-CP

Actin

p53

PARP

FasL

Nutlin-3 (8 �M)- + + + +

Scha

si-

FasL

NO

K-1

cn

trl ab

si-

scr

DM

SO

si-

FasL

NO

K-1

cn

trl ab

si-

scr

DM

SO

c

010

01

28

Eve

nts

110

210310

Fas membrane expression

NCCIT

IgG

DMSO

Nutlin-3 (4�M)

CDDP (4�M)

Nutlin-3 + CDDP

b

p53

Nutlin-3 (�M)

Tera-CP Scha

0 4 80 4 8

si scr

0 4 80 4 8

PARP

Actin

a

dDMSO Nutlin (4�M)

Hoechst

p21

21

02

EP

si p53 si scr si p53

Supplemental Figure 4. (a-b) Survival of TC cells after 96h of continuous Nutlin-3

treatment as indicated, in combination with increasing cisplatin concentration. IC50

values are depicted for cisplatin as well as the combination; values are the mean ± SD.

a b833KE

DMSO

Nutlin-3 (1 �M)

Nutlin-3 (0.5 �M)

0

20

40

60

80

100

Su

rviv

al

(%)

Cisplatin (�M)

0.03130

0.06250.125

0.25 0.5 1 2 4 168

IC50 = 1.04

IC50 = 0.13

0

20

40

60

80

100

Su

rviv

al

(%)

Cisplatin (�M)

0.03130

0.06250.125

0.25 0.5 1 2 4 168

IC50 = 4.05

IC50 = 0.39

DMSO

Nutlin-3 (1 �M)

2102EP NCCIT

DMSO

Nutlin-3 (4 �M)

0

20

40

60

80

100

Su

rviv

al

(%)

Cisplatin (�M)

0

0.06250.125

0.25 0.5 1 2 4 168

IC = 50 2.15

IC = 50 2.25

Supplemental Figure 5. (a) Increased levels of p53 and increased PARP cleavage after targeting the

MDM2/p53 axis; a representative example of three independent experiments is shown. (b) Increased apoptosis

after targeting the MDM2/p53 axis with either siRNA against MDM2 or Nutlin-3 (Nut-3) in combination with

cisplatin. Values are the mean ± SD of three experiments; #p < 0.05; *p < 0.01; **p < 0.005. (c) Following the

indicated treatment TC cells were harvested and Fas membrane expression was determined by flow cytometry.

Values were depicted as mean fluorescence intensity (MFI). Values are the mean ± SD of three experiments.

(d) Decreased apoptotic response after blocking of FasL, with Nok-1, in TC cells treated with the combination

of cisplatin and Nutlin-3. Values are the mean ± SD of three experiments; #p < 0.05; *p < 0.01; **p < 0.005.

0 4 8 0 4 8 Cisplatin (�M)0

20

40

60

80

100

Scha 2102EP

Ap

op

tosis

(%

)

siRNA scrsiRNA Mdm2 Nutlin-3 (2 �M)

2102EPScha

0 0 04 4 48 8 8

si scr si Mdm2 Nut-3 (2 �M)

0 0 04 4 48 8 8

PARP

Mdm2

p53

Cisplatin (�M)

Actin

si scr si Mdm2 Nut-3 (2 �M)

0 4 8 0 4 8 Cisplatin (�M)0

20

40

60

80

100

Scha 2102EP

Ap

op

tosis

(%

)

0 4 8 0 4 8 0 4 8

Scha

0

20

40

60

80

Fas m

em

bra

ne e

xp

ressio

n (

MF

I) siRNA scr

siRNA Mdm2

Nutlin-3 (2 �M)

0 4 8 0 4 8 0 4 8

2102EP

Nutlin-3 (2 �M) + cntrl abNutlin-3 (2 �M) + NOK-1

c

a b

d

**

*

****

#

*** **

****

**

#

Supplemental Materials and Methods

Antibodies

The following antibodies were used: mouse anti p53 (DO-1, Santa Cruz), mouse anti-Mdm2

(SMP14, Oncogene Research Products, Darmstadt, Germany), mouse anti b-Actin (MP Biomedicals,

Eindhoven, the Netherlands), mouse anti p21 (F5, Santa Cruz), rabbit anti-Parp (Roche Diagnostics,

Almere, the Netherlands), caspase 8 (1C12, Cell Signalling, MA, USA), and anti-FasL (C20, Santa Cruz).

RNA interference sequences

Sequence for p53 I small interfering RNA (siRNA) molecules was 5’-GCA UGA ACC GGA GGC CCA

UdTdT-3’ (sense) and 5’-AUG GGC CUC CGG UUC AUG CdTdT-3’ (anti-sense), sequence for p53 II

siRNA was 5’-CUU CGA CUU UGU CAC CGA GdTdT-3’ (sense) and 5’-CUU ACG CUG AGU ACU

UCG AdTdT-3’ (anti-sense), sequence for P21 I siRNA was 5’-GAC CAU GUG GAC CUG UCA CTdT-3’

(sense) and 5’- GUG ACA GGU CCA CAU GGU CdTdT-3’ (anti-sense), sequence for P21 II siRNA was

5’-CUU CGA CUU UGU CAC CGA GTdT-3’ (sense) and 5’-CUC GGU GAC AAA GUC GAA GTdT-3’

(antisense), sequence for FasL siRNA was 5’-CTG GGC TGT ACT TTG TAT AdTdT-3’ (sense) and 5’-

TAT ACA AAG TAC AGC CCA GdTdT-3’ (anti-sense), sequence for MDM2 siRNA was 5’-GTG AAT CTA

CAG GGA CGC CAT CdTdT-3’ (sense) and 5’-GAT GGC GTC CCT GTA GAT TCA CdTdT-3’ (anti-sense).