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:
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
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
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
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.
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.
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.
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.
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.
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.