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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. - PowerPoint PPT Presentation
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Supplemental Figure 1
a
% p
ulld
ow
n f
rom
inp
ut
LNCaP/scrambled-siRNA
Figure 1A, 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
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Enhancer Negative Promoter Enhancer Negative Promoter Enhancer Negative Promoter
IgG AR PHB
No Dox No DHT
No Dox DHT
Dox No DHT
Dox DHT
b
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20No Dox No DHTNo Dox + DHTPHB-RNAi No DHTPHB-RNAi + DHT
Enhancer Negative Promoter0
0.20.40.60.81
1.21.4
Enhancer Negative Promoter
No Dox No DHTNo Dox + DHTPHB-RNAi No DHTPHB-RNAi + DHT
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LNCaP/Luc/PHB-siRNA
KLK2 KLK2
Supplemental Figure 2
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T0 15 30 60 120
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Enhancer Promoter
No Dox+RNAi
Taqman PCR IgG Control
Figure 2A, 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).
Enr
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Time (min) after DHT treatment
Time (min) after DHT treatment
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Time (min) after DHT treatment Time (min) after DHT treatment
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IgG
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Enhancer Promoter
Supplemental Figure 3
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*
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012345
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No dox + RNAi No dox + RNAi
KLK2 TMPRSS2
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Figure 3A. 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.
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pcDNA4-Empty pcDNA4-PHB wt
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nM DHT nM ASD
DHT Androstenedione
Supplemental Figure 4
PS
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DHT concentration (nM) Androstenedione concentration (nM)
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LNCaP/ pcDNA4/TO Empty VectorNo Dox
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DHT concentration (nM) Androstenedione concentration (nM)
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LNCaP/pTER Scrambled Vectorc
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Time after treatment (hrs) Time after treatment (hrs)
DHT Androstenedione
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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.
0 1 2 3 4 50
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Dox
[3H]-Mib (nM)
Specific Binding (cpm)
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[3H]-Mib (nM)
Specific Binding (cpm) 0 1000 2000 3000 40000
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Bound/Free
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Supplemental Figure 5
No Dox + PHB-cDNA
Bmax 2669± 110.9 2635 ± 117.5
Kd 0.70 ± 0.08 0.63 ± 0.08
No Dox + PHB-RNAi
Bmax 3034 ± 107.7 3015 ± 74.53
Kd 0.61 ± 0.06 0.76 ±0.05
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
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-actin TAP1 Cyc D Caspase 7 YY1 TK1
No Dox
PHB RNAi
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TAP1 actin Cyc D PSA
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fold
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)a
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- IFN + IFN - IFN + IFN
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Figure 6.A, Taqman RT-PCR analysis of TAP1, -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
+ Dox (PHB RNAi)
No
DN
ase
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arke
r
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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
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Scrambled PHB-siRNA
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LNCaP VCaP Du145 C42 C42b
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xpre
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f P
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o L
NC
aP)
Cell Line
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.
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’
actin promoter region 1 For 5’-AAGGCAACTTTCGGAACGG-3’Rev 5’-TCCTCTTCCTCAATCTCGCTCTC-3’
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’
Supplemental
Table 1
Table 1: A list of the primer sets used for the ChIP analysis PCR for PSA, KLK2, ß-actin, TAP1 and CyclinD1 gene promoters.
