GAPDH

0

0

CDDP (10 μM)

Epoximicin (nM)

Lactacystine (μM)

0

4

0

8

10

0

20

0

DMSO

0

10

20

30

40

**

Ap

op

tos

is (

% o

f T

ota

l)

38 kDa

80 kDa∆Np73α 70 kDa

1.0 0.5 0.3 0.8 0.4 0.3 0.2 0.1 0.2 0.1 TAp73α/GAPDH∆Np73α/GAPDH 1.0 0.3 0.2 0.4 0.2 0.4 0.3 0.2 0.2 0.1

TAp73α

P53 53 kDa

0

0

0

4

0

8

10

0

20

0

B

Xiap

GAPDH

OV2008

80 kDa

38 kDa

54 kDa

70 kDa∆Np73α

Cleaved-PARP 86 kDaTAp73α

CDDP (µM)

LacZ MOI)

Xiap-s (MOI)

5

0

0

5

0

10

0

5

0

0

5

10

0

10

20

30

40

***

Apo

ptos

is (%

of T

otal

)

5

0

10

A

Figure 1: The CDDP-induced p73α down-regulation is not a consequence of cell-death and was not affected by proteasome inhibition in chemosensitive cells. A) Chemosensitive (OV2008) was infected with adenovirus carrying Xiap-s cDNA or LacZ (5 MOI; 48 h) and then treated with CDDP (10 μM; 24 h). B) OV2008 was pre-treated for thirty minutes with different concentrations of Epoximicin (10 and 20 nM) or Lactacystine (4 and 8 μM) followed by CDDP treatment (10 μM; 24 h). Xiap over-expression, p73α isoforms and GAPDH contents and cleaved α-fodrin and -PARP and apoptosis were assessed by Western blot and Hoechst stain, respectively. Figures represent the mean ± SEM of three independent experiments assessed by two way-ANOVA (**P < 0.01, ***P < 0.001).

0 0.5 1 2.5 5 10

A2780s

5-aza-D(µM)TAp73α

GAPDH 38 kDa

80 kDa

0 0.5 1 5 100

10

20

30

40

a

b b

c c

5-Aza Deoxycitidine (mM)

Apo

ptos

is (%

of t

otal

)

Figure 2: The 5-Aza Deoxycitidine, a demethylating agent, restored TAp73α content in chemosensitive (A2780s) cells. A) Chemosensitive (A2780s) cells were treated with different concentrations of 5-Aza Deoxycitidine (0, 0.5, 1, 2.5, 5 and 10 µM; 3 days). TAp73α and GAPDH contents and apoptosis were detected by Western blot and Hoechst stain, respectively. Results were obtained from three independent experiments using one-way ANOVA (Different letters indicate significant differences).

Time (h)

CDDP (10µM)

Cell Lines

TAp73α∆Np73αGAPDH

A2780s A2780cp

- - + - + - + - - + - + - + 0 6 12 24 0 6 12 24

80 kDa70 kDa38 kDa

0 6 12 18 24

0

10

20

30

A2780s

A2780s+CDDP

A2780cpA2780cp+CDDP

***

***

Time (h)

%

of A

popt

osis

A2780s A2780cp

- + - +

314 bp

533 bp

629 bp

∆Np73α

β-Actin

TAp73α

CDDP (10µM)

Cell line

Figure 3: TAp73α and ΔNp73α protein (A) and mRNA (B) content in chemosensitive (A2780s) and chemoresistant (A2780cp) cells. Both cells were treated with CDDP (10 µM) and harvested at different time point (0, 6, 12 & 24 h). TAp73α and ΔNp73α mRNA and protein content and apoptosis were detected by RT-PCR, Western blot and Hoechst stain, respectively. Figures indicate the average ± SEM of three independent experiments assessed by two way-ANOVA (***P < 0.001).

A

B