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Supplemental Figure 1. 0. LNCaP/Luc/PHB-siRNA. b. No Dox No DHT. No Dox No DHT. No Dox + DHT. *. No Dox + DHT. 20. 1.4. PHB-RNAi No DHT. PHB-RNAi No DHT. *. PHB-RNAi + DHT. PHB-RNAi + DHT. 1.2. *. 15. 1. *. Fold Enrichment of AR. 0.8. Fold Enrichment of PHB. 10. 0.6. 0.4.

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Supplemental Figure 1

0

LNCaP/Luc/PHB-siRNA

b

No Dox No DHT

No Dox No DHT

No Dox + DHT

*

No Dox + DHT

20

1.4

PHB-RNAi No DHT

PHB-RNAi No DHT

*

PHB-RNAi + DHT

PHB-RNAi + DHT

1.2

*

15

1

*

Fold Enrichment of AR

0.8

Fold Enrichment of PHB

10

0.6

0.4

5

0.2

0

0

Enhancer

Negative

Promoter

Enhancer

Negative

Promoter

KLK2

KLK2

a

LNCaP/scrambled-siRNA

60

No Dox No DHT

50

No Dox DHT

Dox No DHT

40

Dox DHT

% pulldown from input

30

20

10

Enhancer

Negative

Promoter

Enhancer

Negative

Promoter

Enhancer

Negative

Promoter

IgG

AR

PHB

Figure 1

A, ChIP analysis of AR and PHB binding to the PSA promoter in the LNCaP/scrambled-siRNA as a percentage of input DNA after treatment with DHT for 0-2hrs (± doxycycline). IgG controls are given for comparison. B, ChIP analysis of AR and PHB binding to the KLK2 promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). * = P<0.05 (t-test analysis).ß


Supplemental Figure 2

Enhancer

Promoter

10

10

8

8

6

6

No Dox

No Dox

+RNAi

+RNAi

4

4

2

2

0

0

T0

15

30

60

120

T0

15

30

60

120

Time after DHT treatment

Time after DHT treatment

b

16

1.2

No Dox

No Dox

PHB RNAi

PHB RNAi

14

1

12

0.8

10

0.6

8

6

0.4

4

0.2

2

0

0

0

15

30

60

120

240

0

15

30

60

120

240

Time (min) after DHT treatment

Time (min) after DHT treatment

1.2

6

No Dox

No Dox

PHB RNAi

PHB RNAi

1

5

0.8

4

PHB enrichment (fold increase)

PHB enrichment (fold increase)

0.6

3

0.4

2

0.2

1

0

0

0

15

30

60

120

240

0

15

30

60

120

240

Time (min) after DHT treatment

Time (min) after DHT treatment

5

5

No Dox

No Dox

PHB RNAi

PHB RNAi

4

4

IgG enrichment (fold increase)

IgG enrichment (fold increase)

3

3

2

2

1

1

0

0

0

15

30

60

120

240

0

15

30

60

120

240

Time (min) after DHT treatment

Time (min) after DHT treatment

Taqman PCR IgG Control

a

Enrichement due to IgG

Enrichement due to IgG

Enhancer Promoter

AR enrichment (fold increase)

AR enrichment (fold increase)

Figure 2

A, ChIP analysis of the PSA promoter and enhancer regions with a control rabbit IgG antibody, in LNCaP/Luc/PHB-siRNA cells treated with DHT over 0-2hours. B, ChIP analysis of AR and PHB binding (and IgG control) to the KLK2 promoter in the LNCaP/Luc/PHB-siRNA cells after treatment with DHT for 0-4hrs (± doxycycline).


Supplemental Figure 3

10

*

9

8

0

7

20

*

6

40

Fold Increase in Expression

5

*

60

4

120

3

240

2

480

1

0

No dox

+ RNAi

No dox

+ RNAi

KLK2

TMPRSS2

a

Time after treatment (min)

b

DHT

Androstenedione

4

4

No Dox

No Dox

PHB-RNAi

PHB-RNAi

3

3

Luciferase expression (foild increase)

Luciferase expression (foild increase)

2

2

1

1

0

0

nM DHT

nM ASD

0

1

10

0

1

10

0

1

10

0

1

10

pcDNA4-Empty

pcDNA4-PHB wt

pcDNA4-Empty

pcDNA4-PHB wt

Figure 3

A. Taqman RT-PCR analysis of KLK2 and TMPRSS2 transcript levels collected at time intervals (0 – 8hr) from starved LNCaP/Luc/PHB-RNAi cells treated with 10nM DHT. ** = P<0.01, * = P<0.05 (t-test analysis). B, AR-mediated luciferase expression from LNCaP/Luc/PHB-siRNA cells treated with DHT or Androstenedione (0-10nM) for 24hrs (± doxycycline), transiently transfected with either empty pcDNA4 or pcDNA expressing PHB-cDNA coding region which is not targetted by PHB-RNAi.


Supplemental Figure 4

a

DHT

Androstenedione

5

No Dox

5

No Dox

Dox

Dox

4

4

3

3

2

2

1

1

0

0

Starved

1

2

4

6

8

16

Starved

1

2

4

6

8

16

Time after treatment (hrs)

Time after treatment (hrs)

LNCaP/ pcDNA4/TO Empty Vector

6

No Dox

6

No Dox

Dox

Dox

5

5

4

4

3

3

2

2

1

1

0

0

0.01

0.1

1

10

100

0.01

0.1

1

10

100

DHT concentration (nM)

Androstenedione concentration (nM)

PSA Fold Increase

PSA Fold Increase

b

PSA Fold Increase

PSA Fold Increase

c

LNCaP/pTER Scrambled Vector

6

6

No Dox

No Dox

Dox

Dox

5

5

4

4

PSA Fold Increase

PSA Fold Increase

3

3

2

2

1

1

0

0

0.01

0.1

1

10

100

0.01

0.1

1

10

100

Androstenedione concentration (nM)

DHT concentration (nM)

Figure 4.

A, Taqman RT-PCR analysis of PSA transcript levels collected at time intervals (0-16hrs) from starved LNCaP/Luc/scrambled-siRNA cells treated with 10nM DHT or androstenedione. B, Taqman RT-PCR analysis of PSA transcripts from starved LNCaP/Luc/pcDNA4/TO-Empty cells treated with 0-100nM DHT or androstenedione. C, Taqman RT-PCR analysis of PSA transcripts from starved LNCaP/Luc/scrambled-siRNA cells treated with 0-100nM DHT or androstenedione.


Supplemental Figure 5

PHB-cDNA

PHB-RNAi

Figure 5.

Scatchard analysis of [3H]-mibolerone binding to the AR in LNCaP/Luc/PHB-cDNA and RNAi cells. Binding maximum (Bmax) and dissociation constant (kd) are given for each cell line in the table.


Supplemental Figure 6

a

25

20

15

Gene expression (fold increase)

Eth

DHT

10

5

0

TAP1

b-actin

Cyc D

PSA

b

No Dox

1.5

PHB RNAi

Gene expression (fold increase)

1

0.5

0

b-actin

TAP1

Cyc D

Caspase 7

YY1

TK1

c

1.6

1.4

1.2

1

TAP1 expression (fold increase)

0.8

0.6

0.4

0.2

0

- gIFN

+ gIFN

- gIFN

+ gIFN

No Dox

PHB RNAi

Figure 6.

A, Taqman RT-PCR analysis of TAP1, b-actin, CyclinD and PSA transcripts from starved LNCaP cells treated with 10nM DHT or ethanol. B, Taqman RT-PCR analysis of b-actin, TAP1, Cyclin D, Caspase 7, YY1, TK transcript levels collected from LNCaP/Luc/PHB-RNAi cells (± doxycycline). C, Taqman RT-PCR analysis of TAP-1 transcripts from LNCaP/Luc/PHB-RNAi cells treated with 100U/ml g-IFN for 6hours.


Supplemental Figure 7

+ DNase

DNA Marker

No DNase

+ Dox (PHB RNAi)

Increased

DNase sensitivity

Figure 7.

Ethidium bromide stained gel electrophoresis showing motility of DNA extracted from LNCaP/Luc/RNAi cells treated with increased amounts of doxycycline for 24hr and subjected to DNase digestion.


Supplemental Figure 8

3

1.2

2.5

1

2

Fold change

0.8

EthOH

Fold change

1.5

DHT

0.6

1

0.4

0.5

0.2

0

0

Scrambled

PHB-siRNA

Scrambled

PHB-siRNA

PSA

PHB

100

% expression of PHB (relative to LNCaP)

50

LNCaP

VCaP

Du145

C42

C42b

Cell Line

a

b

Figure 8.

A, Taqman RT-PCR analysis of PHB transcript levels from LNCaP, VCaP, C42, C42b, Du145 and MCF-7 cells, normalized via absolute quantification against a standard curve generated using purified PHB RNA. B, Taqman RT-PCR analysis of PHB and PSA levels from starved VCaP cells treated with PHB-siRNA for 48hours and treated with DHT for 24hours, normalized to L19. In each case data represent mean of triplicate experiment and are representative of 2 or more independent experiments.


Supplemental

Table 1

PCR primers for ChIP PSA Promoter

Promoter (AREI) FOR 5’-TCTGCCTTTGTCCCCTAGAT-3’

REV 5’-GCTAGCACTTGCTGTTCTGC-3’

Promoter (AREII) FOR 5’-AGGGATCAGGGAGTCTCACA-3’

REV 5’-GCTAGCACTTGCTGTTCTGC-3’

Negative 1 FOR 5’-CTGTGCTTGGAGTTTACCTGA-3’

REV 5’-GCAGAGGTTGCAGTGAGCC-3’

Negative 2 FOR 5’-AGGGTATCACCAGCCCTTCT-3’

REV 5’-GAGGATGTCGGCAGCTCTAC-3’

Enhancer (AREIII) FOR 5’-ACAGACCTACTCTGGAGGAAC-3’

REV 5’-AAGACAGCAACACCTTTTT-3’

Upstream 1 FOR 5’-TTTAGGGCTTCCCAAGATGA-3’

REV 5’-TGTCACCGGGAAAAGAAAAC-3’

Downstream FOR 5’-CTGTGAGTGCCCAACCCTAT-3’

REV 5’-CTGGGGATGCTCATGTTTTTC-3’

Taqman PCR primers for ChIP PSA Promoter

PSA negative For 5’-TCCACTCCAGCTCTAAGATGGT-3’

PSA negative Rev 5’-CAGGTAAACTCCAAGCACAGTGA-3’

PSA negative probe 5’-FAM-CAGAGGTGGATATAGATAATC-3’

PSA promoter For 5’-GTGCATCCAGGGTGATCTAGTAATT-3’

PSA promoter Rev 5’-CACACCCAGAGCTGTGGAA-3’

PSA promoter probe 5’-FAM-CTAGCACTTGCTGTTCTGC-3’

PSA enhancer For 5’-TGACAGTAAACAAATCTGTTGTAAGAGACA-3’

PSA enhancer Rev 5’-AGCAGGCATCCTTGCAAGAT-3’

PSA enhancer probe 5’-FAM-CCAGGCTTGCTTACTGTC-3’

Primers for Other Gene Promoters (ChIP)

KLK2 Enhancer For 5’-TTTATAATTGGGTTGAAAGCAGACCTA-3’

Rev 5’-AGCAGATTTGTTTACTGTTCAGGACA-3’

KLK2 Negative For 5’-TGGGTGATGTGGTTGGATTGG-3’

Rev` 5’-CCCATGATAACCTCAACCAAAACCT-3’

KLK2 Promoter For 5’-GCCTCCAGACTGATCTAGTATGTGT-3’

Rev 5’-CACACCCAGAGCTGTGGAA-3’

b-actin promoter region 1 For 5’-AAGGCAACTTTCGGAACGG-3’

Rev 5’-TCCTCTTCCTCAATCTCGCTCTC-3’

b-actin promoter region 2 For 5’-GAGCTCTTGGAGGGCATGGA-3’

Rev 5’-CTCTACCTCTCAAGCCCAGGT-3’

TAP1 promoter (STAT binding region) For 5’-AACTGGTGCAAGTGGAAAGG-3’

Rev 5’-GCCAGAAGCTCAGCCATTTA-3’

Cyclin D Region A For 5’-CTCCACCTCACCCCCTAAATC-3’

Rev 5’-AGAGCCCAAAAGCCATCC-3’

Cyclin D Region C For 5’-CCGACTGGTCAAGGTAGGAAG-3’

Rev 5’-ACAACCCCTGTGCAAGTTTC-3’

Table 1: A list of the primer sets used for the ChIP analysis PCR for PSA, KLK2, ß-actin, TAP1 and CyclinD1 gene promoters.


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