Characterization of anti-mineralocorticoid receptor antibody (SC-11412) and its use in chromatin immunoprecipitation (ChIP) assay.

<p>[<b>A</b>] <i>Characterization of SC-11412 antibody</i>: The CHOK1 cells were transfected with vector (<b>lane-1</b>) or the vector containing a mineralocorticoid receptor (MR) (<b>lane-2</b>), labeled with ³⁵S-methionine, immunoprecipitated with SC-11412 antibody to MR, resolved on SDS-PAGE, and the gel used to expose X-ray film. [<b>B</b>] <i>Immunoprecipitation of MR-like proteins from rat colonic epithelial cells</i>: Protein extracts of the colonic epithelial cells from an aldosterone rat was immunoprecipitated with the control (<b>lane-1</b>) or the SC-11412 (<b>lane-2</b>) antibody. The immnoprecipitated proteins were resolved on SDS-PAGE and transferred to the nitrocellulose membrane and probed with the SC-11412 antibody. [<b>C</b>] <i>ChIP assay of MR</i>: Numbers on the X-axis represent primer pairs directed to 1 kb blocks starting at approximately 20 kb upstream (number-1) of the Kcnn4 presumed transcript start site (TSS) and extending to 10 kb downstream (number-29) of the presumed TSS (located in block 21). The Y-axis represents fold enrichment in the RT-PCR comparing the specific to the control antibody. Data presented represents mean ± SE of 4 independent experiments using distal colon epithelial cells from aldosterone (<b>closed bars</b>) and normal rats (<b>open bars</b>). The RNA transcript is indicated by an arrow. The presumed promoter region is immediately to the left of the arrow. Exons 1 and 2 are indicated by blocks. Genomic regions cloned in pGL4.23 are indicated by bars and are numbered.</p

Effect of mineralocorticoid receptor (MR) and aldosterone in HEK293T cells expressing Kcnn4 genomic clone.

<p>Regions of the Kcnn4 genes showing positive response in a ChIP assay and indicated by bars in Figure-1C (clone-1, clone-3, clone-5, clone-6, clone-7 and clone-8) were sub-cloned in pGL4.23 and tested for the presence of MR/aldosterone (mineralocorticoid)-responsive enhancers (MRE). The HEK293T cells were transfected with pGL4.23 or pGL4.23 containing Kcnn4 genomic clones. The cells transfected with Kcnn4 genomic clones were also transfected with MR. Luciferase activity was measured in the cells transfected with the Kcnn4 genomic clones with (<b>hatched bars</b>) and without (<b>grey bars</b>) MR transfection. The luciferase activity in the cells transfected with the Kcnn4 genomic clones and MR was also measured in the presence of 10 nM aldosterone (<b>black bars</b>). For clarity, luciferase activity measured in the presence of a plasmid, MR and aldosterone are shown in cells transfected with pGL-4.23, clones-3, -5 and -6. Results presented represent mean ± SE from 3 different experiments. *<i>p</i><0.05– compared to pGL4.23.</p

Sub-localization of mineralocorticoid response elements (MRE) in clone-5 and clone-6.

<p>The clone-5 was sub-cloned into thirds, clone-5A, clone-5B and clone-5C representing the left, middle and right thirds of clone-5, respectively. Clone-5A, -5B and -5C were tested for MRE/aldosterone responsiveness in the HEK293T cells. The HEK293T cells transfected with clone-5A and clone-5B exhibited a significantly higher luciferase activity compared to the pGL4.23 vector transfected cells. Neither clone-5A nor clone-5B contained candidate MREs as analyzed using the Transcription Element Search System (TESS) or MatInspector. Further sub-clones of clone-5B were made (clone-5Ba and clone-5Bb that represent the left and right half of clone-5B, respectively). (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098695#pone.0098695.s001" target="_blank">Figure S1</a> for the sequences). Luciferase activity was not significantly altered in the HEK239T cells transfected with clone-5Ba and clone-5Bb, compared to the cells transfected with a vector (pGL4.23). Clone-6 was also sub-cloned into thirds (clone-6A, clone-6B, and clone-6C). Similar to the clone-5Ba and clone-5Bb transfected cells, luciferase activity was also not significantly altered in the HECK293T cells transfected with clone-6A, clone-6B and clone-6C compared that of cells transfected with a vector (pGL4.23). Data presented represent mean ± SE of triplicate assays of 3 different experiments. *<i>p</i><0.05– compared to pGL4.23.</p

Effect of aldosterone and cortisol concentrations on luciferase activity in mineralocorticoid receptor (MR) and Kcnn4 genomic clones (clone-1, -5 and -6) transfected HEK293T cells.

<p>HEK293T cells were transfected with either MR and a vector (pGL4.23) or MR and the respective Kcnn4 genomic clone (clone-1, clone-5 or clone-6). Increasing concentrations (0–100 nM) of either aldosterone or cortisol were determined on luciferase activity. Clone numbers (1, 5 and 6) are indicated to the right. The pGL4.23 transfected cells were unresponsive to both hormones up to 100 nM (data not shown). Murine Mammary Tumor Virus (MMTV) transfected cells exhibited greater than a 30-fold increase in luciferase activity for both hormones with an EC₅₀ of approximately 1 nM (data not shown). Fitted curves shown were obtained using Graphpad Prism 4 regression analysis software.</p

Sub-localization of mineralocorticoid hormone responsive element (MRE) in clone 1.

<p>Sub-clones of clone-1 were made and tested for MR/aldosterone responsiveness in HEK 293T cells. Clone-1 was sub-cloned into thirds, clone-1A, clone-1B and clone-1C representing the left, middle and right thirds, respectively. Clone-1B and clone-1C exhibited enhanced luciferase activity. Putative MREs were identified in clone-1B and clone-1C using TESS and MatInspector bioinformatics tools. The sequence <u>TCTTGA</u>GTG<u>TGTTCT</u> in clone-1B (candidate MRE underlined) was mutated to <u>TCTTGc</u>GTG<u>cacgac (clone-1B_mut)</u> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098695#pone.0098695.s001" target="_blank">Figure S1</a> for sequence). The candidate MRE <u>GGCTCT</u>GCG<u>TGTTCT</u> in clone-1C was mutated to <u>GGCTgT</u>GCG<u>cacgac (clone-1C_mut).</u> The absence of the enhanced luciferase activity in the HEK293T cells transfected with c<u>lone-1B_mut</u> and <u>clone-1C_mut</u> showed a loss of function, thus demonstrating functionality of the MREs localized in clone-1B and clone-1C. Data presented represent mean ± SE of triplicate assays from 3 different experiments. *<i>p</i><0.05– compared to pGL4.23.</p

Functional activity of clone-1, clone-5 and clone-6 expressed in CaCo2 cells.

<p>The CaCo-2 cells were transfected with either a vector (pGL4.23), clone-1, clone-5, or clone-6. After the transfection, the luciferase activity was measured in the presence and absence of aldosterone (10 nM) treatment. Relative luciferase activity was normalized to the luciferase activity present in the vector transfected cell (pGL4.23; considered 1.0). Data presented represent mean ± SE, from triplicate assays from 3 different experiments). *<i>p</i><0.05– compared to pGL4.23.</p

Torasemide does not act as a MR antagonist for the regulation of endogenous genes in H9C2-MR cells in the presence of 10⁻⁸ M aldosterone.

<p>10⁻⁸ M aldosterone (Aldo) increased expression of the aldosterone-targets genes Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3 and Tenascin-X. Addition of increasing doses of spironolactone (A+S) inhibited aldosterone-induced gene expression. In contrast, increasing concentrations of torasemide (A+T) had no antagonistic effect. Mean ± SEM (n = 4). *<i>p</i><0.05 <i>vs</i> control (Ctrl); # <i>p</i><0.05 <i>vs</i> aldosterone.</p

In vivo kinetics of nuclear translocation of MR in COS-7 cells.

<p>COS-7 cells transfected with GFP-MR were treated with 10⁻⁸ M aldosterone (Aldo), 10⁻⁸ M aldosterone +10⁻⁶ M torasemide (Aldo+ Tora), 10⁻⁸ M aldosterone +10⁻⁶ M spironolactone (Aldo+Spiro), 10⁻⁶ M spironolactone (Spiro) or 10⁻⁶ M torasemide (Tora), starting at time 0. Individual points represent the average percentage fluorescent intensity of the nucleus vs. total cellular fluorescence (Fn/Ft) measured in individual cells over the indicated period of time (± SE, n = 16). Data points were fitted to a sigmoid curve.</p

Torasemide does not act as a MR antagonist for the regulation of endogenous genes in H9C2-MR cells in the presence of 10⁻⁹ M aldosterone.

<p>10⁻⁹ M aldosterone (Aldo) increased expression of the aldosterone-targets genes Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3 and Tenascin-X. Addition of increasing doses of spironolactone (A+S) inhibited aldosterone-induced gene expression. In contrast, increasing concentrations of torasemide (A+T) had no antagonistic effect. Mean ± SEM (n = 4). *<i>p</i><0.05 <i>vs</i> control (Ctrl); # <i>p</i><0.05 <i>vs</i> aldosterone.</p