Supplementary Figure 1 ACBED - cloudfront.net
Supplementary Figure 1 C
C
F H -7 C C M 193 D A- 7 T4 46 7D 8 H C C H 114 C C 3 SU 38 M 14 9
A
TAp73_ TAp73β
B
Relative TAp73 mRNA
p73
250 200 150
TAp73 mRNA Levels
M
300 2
R = 0.9121
250
MDA-468
200
HCC1143
150 MCF-7
100 SUM149
1
50
HCC1937
HCC38
50 0
100
T47D
10
100
TAp73 Protein levels
M
D p63 _-tub.
ΔNp63_
E RelativeΔNp63 mRNA mRNA Levels Relative
C S U 38 M 14 9
43
C
H
11
7D H
C
C
8
T4
46
37
A-
H 7 C C M -19 D A- 37 T4 MB 7D -4 68
C
F-
H
M
D
19
C C
M
C
F-
7
0
18 16 14 12 10 8 6 4 2 0 MCF-7
HCC1937 MDA-468
T47D
1.0 0.8 0.6 0.4 0.2 0.0 Vector
p63si
MCF-7 p63si
12 10 8 6 4 2
FA S
Ba
x p5 3A IP 1
0 NO XA
Vector
PU M A
B
1.2
Fold Change
A
Relative ΔNp63 mRNA Relative DNp63 mRNA Levels
Supplementary Figure 2
Supplementary Figure 3
T47D
Vector TAp73si
Vector
V
TAp73si
p63
80
TAp73_ TAp73b
p73
60 40
PARP
20 0
C
Vector
100
TAp73
Vector
PUMA ΔNp73
p63si
_-tubulin
D % Apoptosis
Relative mRNA Levels
120
p6 3s i
B
p6 3s i V
A
40 Vector p63si
30 20 10
TAp73si
0 Vector
TAp63si
TAp73si
Supplementary Figure 4 A
i i i tor p73s ctor p73s ctor p73s c IP p73 Ve TA Ve TA Ve TA
TAp73_ TAp73β
p73 MCF-7 HCC1937 MDA-468
B
Cell Lines Cisplatin (uM) Doxorubicin (nM) Paclitaxel (nM) MCF-7 Vector 6.00 ± 0.57 < 10.00 0.59 ± 0.04 TAp73si 9.22 ± 0.19 < 10.00 0.59 ± 0.06 HCC1937 Vector 2.80 ± 1.48 40.14 ± 10.5 0.78 ± 0.08 TAp73si 43.75 ± 0.59 20.31 ± 9.26 0.53 ±0.50 MDA-MB-468 Vector 1.15 ± 0.40 38.43 ± 7.70 0.56 ± 0.02 TAp73si 12.86 ± 3.10 69.28 ± 2.76 0.64 ± 0.06
120
C % Viability
100 80 60 40
Vector TAp73si Allp73si
20 0 0.01
D p73
MCF-10A r 3_ cto Ap7 Ve T
E
0.1 1 10 Cisplatin (µ M)
100
MCF-10A Cisplatin (µM) Doxorubicin (nM) Paclitaxel (nM) Vector 6.06 ± 0.55 57.44 ± 2.73 8.23 ± 1.26 TAp73b 0.74 ± 0.03 73.29 ± 6.88 8.12 ± 0.15
Supplementary Figure Legends Supplementary Figure 1 Correlation between p73 and p63 mRNA and protein levels in breast cancer cells. (A) TAp73 protein level by IP/Immunoblot of 2.5mg protein lysate from the indicated human breast cancer-derived cell lines. Little or no ΔNp73 is detected based on transfected isoform size controls and on isoform-specific QRT-PCR (not shown). (B) Quantitation of TAp73 by isoform-specific QRT-PCR, normalized to GAPDH. (C) Correlation of TAp73 mRNA and protein levels in breast cancer cells. (D) Expression of ΔNp63α protein by immunoblot. (E) Expression of ΔNp63 mRNA by isoform-specific QRT-PCR in the indicated cell lines. Error bars in B and E show SD for two experiments. Supplementary Figure 2 Controls for apoptosis following p63 inhibition by lentiviral shRNA. (A) Knockdown of endogenous ΔNp63 mRNA by lentiviral p63-directed shRNA (p63si) in HCC-1937. RNA was prepared at 72 hours following lentiviral infection and assayed by isoform-specific QRT-PCR, normalized to GAPDH levels. (B) Neither gene induction nor apoptosis is observed in p63-negative MCF-7 cells. Left, photomicrographs (100X) taken at 72 hours post lentiviral shRNA infection. Right, QRTPCR for the indicated genes. For gene expression graphs, error bars represent SD from two independent experiments performed in duplicate. Supplementary Figure 3 Puma induction, PARP cleavage, and apoptosis following p63 knockdown are TAp73-dependent in T47D cells. (A) TAp73-directed shRNA targets TAp73 but not ΔNp73. Expression of endogenous TAp73 and ΔNp73 was assessed by QRT-PCR following lentiviral shRNA infection. Error bars indicate SD from three measurements. (B) Cells expressing a TAp73-directed shRNA or control were subsequently infected with a p63-directed lentiviral shRNA and harvested at 72 hours for immunoblot and IP/immunoblot (for p73). (C) Photomicrograph (100X) taken 72 hours post lentiviral infection as in B show that morphologic features of apoptosis correlate with Puma induction and PARP cleavage. (D) Rescue from apoptosis following ablation of TAp73 but not TAp63. Quantitation of apoptosis by Annexin/PI staining of cells
1
treated as in B and harvested 72 hours following p63 knockdown. Error bars show SD for 3 independent experiments.
Supplementary Figure 4 TAp73 mediates cisplatin sensitivity in breast cancer cells. (A) Knockdown of endogenous TAp73 protein by lentiviral shRNA in the indicated cell lines, assessed by IP/immunoblot. (B) IC50 values determined by cell viability (MTT) assay 5 days following treatment of control or TAp73-directed shRNA-expressing cells with the indicated agents. Values indicate the mean + SD for 3 independent experiments. (C) Knockdown of TAp73 or All p73 isoforms induces cisplatin resistance in breast cancer cells. MTT assay performed as in B. (D) Stable retroviral expression of TAp73β in MCF-10A cells, assessed by immunoblot. (E) TAp73β expression in mammary epithelial cells conveys specific sensitivity to cisplatin. IC50 values determined and expressed as in B.
2
Supplementary Table 1. p53 Status in Clinical Breast Cancer Samples Sample ID ER Status P53 Status 282 432 481 480 398 298 469 436 477 426 430 242 356 253 588 1274
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative
WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT Mutant
164 720 209 1196 465 1322 1278 1106 222 1321 715 999 158 489 1502 289 1325 1273 756 1509 488
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
Mutant Mutant Mutant Mutant Mutant WT WT WT WT WT Mutant WT WT Mutant WT WT WT Mutant WT WT WT
Codon 72 Codon nucleotide polymorphism Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Unknown Pro 151 (G to T) 152 (A to G) Arg 523 (C to G) Arg 639 (A to G) Arg 818 (G to A) Pro 730 (G to A) Arg Del (529-546) Arg Arg Arg Unknown Arg Pro 577 (C to T) Arg Arg Pro 469 (G to T) Arg Pro Pro Pro 817 (C to T) Arg Pro Pro
Codon Amino Acid
Stop E51G R175G R213R R273H G244S
H193Y V157F
R273C
Mutational status was determined based on bi-directional sequencing (exon 1-7), and was verified by the presence of nucleotide changes in both strands.
Supplementary Table 2. Primer sequences for QRT-PCR Gene
Forward primer
Reverse primer
Product (bp)
Δ Np63
5’-GGAAAACAATGCCCAGACTC- 3’
5’-GTGGAATACGTCCAGGTGGC -3’
294
TAp63
5’-AAGATGGTGCGACAAACAAG- 3’
5’-AGAGAGCATCGAAGGTGGAG -3’
234
TAp73
5'-GCACCACGTTTGAGCACCTCT -3'
5’-GCAGATTGAACTGGGCCATGA - 3'
168
Δ Np73
5'-CAAACGGCCCGCATGTTCCC- 3'
5'-TTGAACTGGGCCGTGGCGAG -3'
232
Bax
5’-TGACATGTTTTCTGACGGCAAC-3’
5’-GGAGGCTTGAGGAGTCTCAC C- 3’
204
Noxa
5’-GAGATGCCTGGGAAGAAGG-3’
5’-ACGTGCACCTCCTGAGAAAA -3’
228
P53AIP1
5’-AGCTCACTCCGAAAGCCTCTGCTC- 3’
5’-GCATCACCGAGAGGTTCTGG TCTC-3’
280
FAS
5’-GGGCATCTGGACCCTCCTAC- 3’
5’-TCCTTTCACCTGGAGGACAG -3’
200
PUMA
5’-ACGACCTCAACGCACAGTACGAG-3’
5’-AGGAGTCCGCATCTCCGTCA GTG-3’
345
HER2
5’-AGCGGTGTGAAACCTGACC-3’
5’-TTGATGAGGATCCCAAAGACC - 3’
224
B2M
5’- AGCTGTGCTCGCGCTACTCT C-3’
5’-CACACGGCAGGCATACTCAT C- 3’
286
GAPDH
5’-CACCCAGAAGACTGTGGATGG -3’
5’-GTCTACATGGCAACTGTGAG G- 3’
587
C
F H -7 C C M 193 D A- 7 T4 46 7D 8 H C C H 114 C C 3 SU 38 M 14 9
A
TAp73_ TAp73β
B
Relative TAp73 mRNA
p73
250 200 150
TAp73 mRNA Levels
M
300 2
R = 0.9121
250
MDA-468
200
HCC1143
150 MCF-7
100 SUM149
1
50
HCC1937
HCC38
50 0
100
T47D
10
100
TAp73 Protein levels
M
D p63 _-tub.
ΔNp63_
E RelativeΔNp63 mRNA mRNA Levels Relative
C S U 38 M 14 9
43
C
H
11
7D H
C
C
8
T4
46
37
A-
H 7 C C M -19 D A- 37 T4 MB 7D -4 68
C
F-
H
M
D
19
C C
M
C
F-
7
0
18 16 14 12 10 8 6 4 2 0 MCF-7
HCC1937 MDA-468
T47D
1.0 0.8 0.6 0.4 0.2 0.0 Vector
p63si
MCF-7 p63si
12 10 8 6 4 2
FA S
Ba
x p5 3A IP 1
0 NO XA
Vector
PU M A
B
1.2
Fold Change
A
Relative ΔNp63 mRNA Relative DNp63 mRNA Levels
Supplementary Figure 2
Supplementary Figure 3
T47D
Vector TAp73si
Vector
V
TAp73si
p63
80
TAp73_ TAp73b
p73
60 40
PARP
20 0
C
Vector
100
TAp73
Vector
PUMA ΔNp73
p63si
_-tubulin
D % Apoptosis
Relative mRNA Levels
120
p6 3s i
B
p6 3s i V
A
40 Vector p63si
30 20 10
TAp73si
0 Vector
TAp63si
TAp73si
Supplementary Figure 4 A
i i i tor p73s ctor p73s ctor p73s c IP p73 Ve TA Ve TA Ve TA
TAp73_ TAp73β
p73 MCF-7 HCC1937 MDA-468
B
Cell Lines Cisplatin (uM) Doxorubicin (nM) Paclitaxel (nM) MCF-7 Vector 6.00 ± 0.57 < 10.00 0.59 ± 0.04 TAp73si 9.22 ± 0.19 < 10.00 0.59 ± 0.06 HCC1937 Vector 2.80 ± 1.48 40.14 ± 10.5 0.78 ± 0.08 TAp73si 43.75 ± 0.59 20.31 ± 9.26 0.53 ±0.50 MDA-MB-468 Vector 1.15 ± 0.40 38.43 ± 7.70 0.56 ± 0.02 TAp73si 12.86 ± 3.10 69.28 ± 2.76 0.64 ± 0.06
120
C % Viability
100 80 60 40
Vector TAp73si Allp73si
20 0 0.01
D p73
MCF-10A r 3_ cto Ap7 Ve T
E
0.1 1 10 Cisplatin (µ M)
100
MCF-10A Cisplatin (µM) Doxorubicin (nM) Paclitaxel (nM) Vector 6.06 ± 0.55 57.44 ± 2.73 8.23 ± 1.26 TAp73b 0.74 ± 0.03 73.29 ± 6.88 8.12 ± 0.15
Supplementary Figure Legends Supplementary Figure 1 Correlation between p73 and p63 mRNA and protein levels in breast cancer cells. (A) TAp73 protein level by IP/Immunoblot of 2.5mg protein lysate from the indicated human breast cancer-derived cell lines. Little or no ΔNp73 is detected based on transfected isoform size controls and on isoform-specific QRT-PCR (not shown). (B) Quantitation of TAp73 by isoform-specific QRT-PCR, normalized to GAPDH. (C) Correlation of TAp73 mRNA and protein levels in breast cancer cells. (D) Expression of ΔNp63α protein by immunoblot. (E) Expression of ΔNp63 mRNA by isoform-specific QRT-PCR in the indicated cell lines. Error bars in B and E show SD for two experiments. Supplementary Figure 2 Controls for apoptosis following p63 inhibition by lentiviral shRNA. (A) Knockdown of endogenous ΔNp63 mRNA by lentiviral p63-directed shRNA (p63si) in HCC-1937. RNA was prepared at 72 hours following lentiviral infection and assayed by isoform-specific QRT-PCR, normalized to GAPDH levels. (B) Neither gene induction nor apoptosis is observed in p63-negative MCF-7 cells. Left, photomicrographs (100X) taken at 72 hours post lentiviral shRNA infection. Right, QRTPCR for the indicated genes. For gene expression graphs, error bars represent SD from two independent experiments performed in duplicate. Supplementary Figure 3 Puma induction, PARP cleavage, and apoptosis following p63 knockdown are TAp73-dependent in T47D cells. (A) TAp73-directed shRNA targets TAp73 but not ΔNp73. Expression of endogenous TAp73 and ΔNp73 was assessed by QRT-PCR following lentiviral shRNA infection. Error bars indicate SD from three measurements. (B) Cells expressing a TAp73-directed shRNA or control were subsequently infected with a p63-directed lentiviral shRNA and harvested at 72 hours for immunoblot and IP/immunoblot (for p73). (C) Photomicrograph (100X) taken 72 hours post lentiviral infection as in B show that morphologic features of apoptosis correlate with Puma induction and PARP cleavage. (D) Rescue from apoptosis following ablation of TAp73 but not TAp63. Quantitation of apoptosis by Annexin/PI staining of cells
1
treated as in B and harvested 72 hours following p63 knockdown. Error bars show SD for 3 independent experiments.
Supplementary Figure 4 TAp73 mediates cisplatin sensitivity in breast cancer cells. (A) Knockdown of endogenous TAp73 protein by lentiviral shRNA in the indicated cell lines, assessed by IP/immunoblot. (B) IC50 values determined by cell viability (MTT) assay 5 days following treatment of control or TAp73-directed shRNA-expressing cells with the indicated agents. Values indicate the mean + SD for 3 independent experiments. (C) Knockdown of TAp73 or All p73 isoforms induces cisplatin resistance in breast cancer cells. MTT assay performed as in B. (D) Stable retroviral expression of TAp73β in MCF-10A cells, assessed by immunoblot. (E) TAp73β expression in mammary epithelial cells conveys specific sensitivity to cisplatin. IC50 values determined and expressed as in B.
2
Supplementary Table 1. p53 Status in Clinical Breast Cancer Samples Sample ID ER Status P53 Status 282 432 481 480 398 298 469 436 477 426 430 242 356 253 588 1274
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative
WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT Mutant
164 720 209 1196 465 1322 1278 1106 222 1321 715 999 158 489 1502 289 1325 1273 756 1509 488
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
Mutant Mutant Mutant Mutant Mutant WT WT WT WT WT Mutant WT WT Mutant WT WT WT Mutant WT WT WT
Codon 72 Codon nucleotide polymorphism Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Unknown Pro 151 (G to T) 152 (A to G) Arg 523 (C to G) Arg 639 (A to G) Arg 818 (G to A) Pro 730 (G to A) Arg Del (529-546) Arg Arg Arg Unknown Arg Pro 577 (C to T) Arg Arg Pro 469 (G to T) Arg Pro Pro Pro 817 (C to T) Arg Pro Pro
Codon Amino Acid
Stop E51G R175G R213R R273H G244S
H193Y V157F
R273C
Mutational status was determined based on bi-directional sequencing (exon 1-7), and was verified by the presence of nucleotide changes in both strands.
Supplementary Table 2. Primer sequences for QRT-PCR Gene
Forward primer
Reverse primer
Product (bp)
Δ Np63
5’-GGAAAACAATGCCCAGACTC- 3’
5’-GTGGAATACGTCCAGGTGGC -3’
294
TAp63
5’-AAGATGGTGCGACAAACAAG- 3’
5’-AGAGAGCATCGAAGGTGGAG -3’
234
TAp73
5'-GCACCACGTTTGAGCACCTCT -3'
5’-GCAGATTGAACTGGGCCATGA - 3'
168
Δ Np73
5'-CAAACGGCCCGCATGTTCCC- 3'
5'-TTGAACTGGGCCGTGGCGAG -3'
232
Bax
5’-TGACATGTTTTCTGACGGCAAC-3’
5’-GGAGGCTTGAGGAGTCTCAC C- 3’
204
Noxa
5’-GAGATGCCTGGGAAGAAGG-3’
5’-ACGTGCACCTCCTGAGAAAA -3’
228
P53AIP1
5’-AGCTCACTCCGAAAGCCTCTGCTC- 3’
5’-GCATCACCGAGAGGTTCTGG TCTC-3’
280
FAS
5’-GGGCATCTGGACCCTCCTAC- 3’
5’-TCCTTTCACCTGGAGGACAG -3’
200
PUMA
5’-ACGACCTCAACGCACAGTACGAG-3’
5’-AGGAGTCCGCATCTCCGTCA GTG-3’
345
HER2
5’-AGCGGTGTGAAACCTGACC-3’
5’-TTGATGAGGATCCCAAAGACC - 3’
224
B2M
5’- AGCTGTGCTCGCGCTACTCT C-3’
5’-CACACGGCAGGCATACTCAT C- 3’
286
GAPDH
5’-CACCCAGAAGACTGTGGATGG -3’
5’-GTCTACATGGCAACTGTGAG G- 3’
587
