Androgen Modulation of Coregulator Expression in Prostate Cancer Cells
Mol Endocrinol Heemers et al. 23: 572 Supplemental Data
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure S1 - Heatmap representing results obtained for androgen-regulated positive control genes. LNCaP cells were seeded in medium supplemented with CSS. Three days later, medium was changed and cells were treated with 0, 0.1 or 1 nM of R1881. 48 hours later, cells were harvested, RNA was isolated and subjected to DASL analysis. Heatmap represents the results from individual samples. 3 biological replicates were used per treatment group (0.0, 0.1 or 1.0 nM of R1881). Genes are listed according to function. Abbreviations used : SECR, secretory function; PA SYN, polyamine synthesis; CYTOSKEL, cytoskeleton; PROL, proliferation. Color intensity shown in each heatmap corresponds to the relative level of expression for a gene across all nine samples. For each gene yellow indicates high expression, red indicates low expression. Genes are listed in line with the order of the genes in Table 1.
- Supplemental Figure S2 - Heatmap representing results obtained for coregulator genes. LNCaP cells were seeded in medium supplemented with CSS. Three days later, medium was changed and cells were treated with 0, 0.1 or 1 nM of R1881. 48 hours later, cells were harvested, RNA was isolated and subjected to DASL analysis. Heatmap represents the results from individual samples. 3 biological replicates were used per treatment group (0.0, 0.1 or 1.0 nM of R1881). Genes are listed according to function. Abbreviations used : CHROM, chromatin remodeling; HAT, histone (de)acetylation; HMT, histone (de)methylation; UB/PROT, ubiquitination/proteasome; SUMO, sumoylation; RNA MET, RNA metabolism; DNA, DNA repair; CH, chaperone; CYTO, cytoskeleton; EN, endocytosis; INT/TRANS, signal integrators and transducers; CYCL, cell cycle. Color intensity shown in each heatmap corresponds to the relative level of expression for a gene across all nine samples. For each gene yellow indicates high expression, red indicates low expression. Arrows indicate a subset of genes selected for follow-up studies. Genes are listed in line with the order of the genes in Table 1.
- Supplemental Figure S3 - Validation of primers used for real-time RT-PCR. LNCaP cells were transfected with specific siRNAs targeting coregulator expression (black bars) or control siRNAs (grey bars). 12-16 hours after transfection, medium was changed. 96 hours later, RNA was isolated and converted into cDNA. Real time RT-PCR was performed with the primer pairs listed in Table S1. Coregulator mRNA levels were normalized with the values obtained from GAPDH. Values are expressed as relative expression levels, taking the value obtained from the control transfected samples as 1. Columns; means of duplicate measurements, bars; SEM. B.
- Supplemental Figure S4 - Effect of the antiandrogen bicalutamide on androgen regulation of coregulator expression. LNCaP cells were seeded in medium supplemented with CSS. Two days later, medium was changed and cells were treated with 1 nM R1881 (black bars) or ethanol vehicle (grey bars) for 48 hours, in the presence or absence of 10μM bicalutamide (Casodex). RNA was isolated, converted into cDNA and real time RT-PCR was performed with. Coregulator mRNA levels were normalized with the values obtained from GAPDH. Values are expressed as relative expression levels, taking the value obtained from one of the untreated samples as 1. Columns, means of values obtained from three independent biological replicates ; bars, SEM.
- Supplemental Figure S5 - Validation of antibodies used for western blotting. LNCaP cells were transfected with specific siRNAs targeting coregulator expression or control (c) siRNAs. 12-16 hours after transfection, medium was changed. 96 hours later, proteins were isolated and western blotting was performed. To assess potential inter-sample loading differences, blots were stripped and reprobed with antibodies recognizing beta-actin (b-act).
- Supplemental Figure S6 - Loss of coregulator expression does not cause apoptosis. LNCaP cells were transfected with specific siRNAs targeting coregulator expression or control (c) siRNAs. 12-16 hours after transfection, medium was changed. 96 hours later, proteins were isolated and western blotting was performed using an antibody directed against PARP. To assess potential inter-sample loading differences, blots were stripped and reprobed with antibodies recognizing beta-actin (b-act). Lower right panel : to verify the assay’s ability to detect PARP cleavage, LNCaP cells were treated with 0, 1 or 5 ng/ml staurosporine for 24 hours. Cell lysates were prepared and western blotting was performed as described.
- Supplemental Table 2 - Effect of androgen treatment on DASL expression profile. LNCaP cells were seeded in medium supplemented with CSS. Three days later, medium was changed and cells were treated with 0, 0.1 or 1 nM of R1881. 48 hours later, cells were harvested, RNA was isolated and subjected to DASL analysis. For each treatment group, the average of the absolute expression values obtained from 3 biological replicates is shown. Standard deviations and statistical significance are listed.
- Supplemental Table 3 - Effect of androgen treatment on DASL expression profile. LNCaP cells were seeded in medium supplemented with CSS. Three days later, medium was changed and cells were treated with 0, 0.1 or 1 nM of R1881. 48 hours later, cells were harvested, RNA was isolated and subjected to DASL analysis. Values represent the average fold change in gene expression following treatment with 0.1 or 1 nM of R1881 from 3 biological replicates each. *, coregulator that has been shown to interact with the AR. Iso, coregulator isoform. Genes are listed in alphabetical order.
- Supplemental Table 1 - Data from Results Section of Manuscript