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This study examines the binding of androgen receptor (AR) and prohibitin (PHB) to promoters and enhancers in LNCaP cells using chromatin immunoprecipitation (ChIP) analysis. The effects of DHT and PHB knockdown on gene expression are also investigated.
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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.