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Supplemental Figure 1 a % pulldown from input LNCaP/scrambled-siRNA Figure 1 A, ChIP analysis of AR and PHB binding to the PSA promoter in the LNCaP/scrambled-siRNA.

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Presentation on theme: "Supplemental Figure 1 a % pulldown from input LNCaP/scrambled-siRNA Figure 1 A, ChIP analysis of AR and PHB binding to the PSA promoter in the LNCaP/scrambled-siRNA."— Presentation transcript:

1 Supplemental Figure 1 a % pulldown from input LNCaP/scrambled-siRNA 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 EnhancerNegativePromoterEnhancerNegativePromoterEnhancerNegativePromoter IgGARPHB No Dox No DHT No Dox DHT Dox No DHT Dox DHT

2 Supplemental Figure 2 a T No Dox +RNAi T Time after DHT treatment Enhancer Promoter No Dox +RNAi Taqman PCR IgG Control 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). Enrichement due to IgG AR enrichment (fold increase) Enhancer Promoter

3 Supplemental Figure 3 a b * * * No dox+ RNAiNo dox+ RNAi KLK2TMPRSS2 Fold Increase in Expression Time after treatment (min) 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 pcDNA4-EmptypcDNA4-PHB wt No Dox PHB-RNAi pcDNA4-EmptypcDNA4-PHB wt No Dox PHB-RNAi Luciferase expression (foild increase) nM DHT nM ASD DHT Androstenedione

4 Supplemental Figure 4 PSA Fold Increase b DHT concentration (nM) Androstenedione concentration (nM) PSA Fold Increase No Dox Dox No Dox Dox LNCaP/pTER Scrambled Vector c PSA Fold Increase 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.

5 Supplemental Figure 5 No Dox+ PHB-cDNA Bmax2669± ± Kd0.70 ± ± 0.08 No Dox + PHB-RNAi Bmax3034 ± ± Kd0.61 ± ±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.

6 Supplemental Figure actin TAP1Cyc DCaspase 7YY1TK1 No Dox PHB RNAi Gene expression (fold increase) TAP1 actin Cyc DPSA Eth DHT Gene expression (fold increase) a IFN+ IFN- IFN+ IFN No DoxPHB RNAi TAP1 expression (fold increase) c b 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.

7 Supplemental Figure 7 + Dox (PHB RNAi) No DNase DNA Marker + DNase 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.

8 Supplemental Figure Scrambled PHB-siRNA PHB Scrambled PHB-siRNA PSA EthOH DHT Fold change b a LNCaPVCaPDu145C42C42b % expression of PHB (relative to LNCaP) 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.

9 PCR primers for ChIP PSA Promoter Promoter (AREI)FOR5-TCTGCCTTTGTCCCCTAGAT-3 REV5-GCTAGCACTTGCTGTTCTGC-3 Promoter (AREII)FOR5-AGGGATCAGGGAGTCTCACA-3 REV5-GCTAGCACTTGCTGTTCTGC-3 Negative 1FOR5-CTGTGCTTGGAGTTTACCTGA-3 REV5-GCAGAGGTTGCAGTGAGCC-3 Negative 2FOR5-AGGGTATCACCAGCCCTTCT-3 REV5-GAGGATGTCGGCAGCTCTAC-3 Enhancer (AREIII)FOR5-ACAGACCTACTCTGGAGGAAC-3 REV5-AAGACAGCAACACCTTTTT-3 Upstream 1FOR5-TTTAGGGCTTCCCAAGATGA-3 REV5-TGTCACCGGGAAAAGAAAAC-3 Downstream FOR5-CTGTGAGTGCCCAACCCTAT-3 REV5-CTGGGGATGCTCATGTTTTTC-3 Taqman PCR primers for ChIP PSA Promoter PSA negative For5-TCCACTCCAGCTCTAAGATGGT-3 PSA negative Rev5-CAGGTAAACTCCAAGCACAGTGA-3 PSA negative probe 5-FAM-CAGAGGTGGATATAGATAATC-3 PSA promoter For5-GTGCATCCAGGGTGATCTAGTAATT-3 PSA promoter Rev5-CACACCCAGAGCTGTGGAA-3 PSA promoter probe 5-FAM-CTAGCACTTGCTGTTCTGC-3 PSA enhancer For5-TGACAGTAAACAAATCTGTTGTAAGAGACA-3 PSA enhancer Rev5-AGCAGGCATCCTTGCAAGAT-3 PSA enhancer probe 5-FAM-CCAGGCTTGCTTACTGTC-3 Primers for Other Gene Promoters (ChIP) KLK2 Enhancer For5-TTTATAATTGGGTTGAAAGCAGACCTA-3 Rev5-AGCAGATTTGTTTACTGTTCAGGACA-3 KLK2 NegativeFor5-TGGGTGATGTGGTTGGATTGG-3 Rev`5-CCCATGATAACCTCAACCAAAACCT-3 KLK2 PromoterFor5-GCCTCCAGACTGATCTAGTATGTGT-3 Rev5-CACACCCAGAGCTGTGGAA-3 actin promoter region 1For5-AAGGCAACTTTCGGAACGG-3 Rev5-TCCTCTTCCTCAATCTCGCTCTC-3 actin promoter region 2For5-GAGCTCTTGGAGGGCATGGA-3 Rev5-CTCTACCTCTCAAGCCCAGGT-3 TAP1 promoter (STAT binding region)For5-AACTGGTGCAAGTGGAAAGG-3 Rev5-GCCAGAAGCTCAGCCATTTA-3 Cyclin D Region AFor5-CTCCACCTCACCCCCTAAATC-3 Rev5-AGAGCCCAAAAGCCATCC-3 Cyclin D Region CFor5-CCGACTGGTCAAGGTAGGAAG-3 Rev5-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.


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