BRCA1 methylation status alters protein-protein interactions at the 504-802 region.

<p>(<b>a</b>) Schematic of BRCA1 504-802 primary sequence depicting important protein-protein interactions and domains that could be affected by the methylation of this region. (<b>b</b>) MDA-MB-231 cells were treated with AdOx (30 µM) in order to observe BRCA1 methylation inhibition upon treatment. Two milligram of MDA-MB-231 whole cell protein extract was immunoprecipitated with anti-BRCA1 or anti-IgG antibodies, separated on a 4-20% gel by SDS-PAGE, and western blotted using antibodies against Sp1 and BRCA1 proteins. Input represents 1/10 of immunoprecipitated material. Results are representative of two independent experiments. (<b>c</b>) MDA-MB-231 cells were treated with AdOx (30 µM) and whole cell extract separated on a 4-20% gel by SDS-PAGE, and probed with anti-Sp1 antibody. Densitometry was averaged from three independent immunoblots.</p

Decreased levels of PRMT1 alters BRCA1 promoter binding <i>in vivo.</i>

<p>(<b>a</b>) HeLa cells were transfected with different concentrations of PRMT1 siRNA (10, 25, 50 nM) following manufacturer's instructions. Results are representative of two independent experiments. (<b>b</b>) HeLa cells transfected with 50 nM Luc or PRMT1 siRNA were collected for ChIP analysis. Anti-BRCA1 (10 µg), anti-IgG (10 µg), and anti-histone H3-phosphorylated at S10 (H3-pS10, 5 µg) antibodies were used for ChIP analysis. PCR products were run on a 2% agarose gel and visualized with ethidium bromide staining. Results are representative of two independent experiments.</p

Subunit expression of cardiac L-VDCC subunits in human myocardial specimens

<p><b>(a)</b> Human specimens from non-failing (NF) and failing (F) myocardium (n = 4–5) were analyzed in immunoblots using specific polyclonal antibodies directed against the particular L-VDCC subunits. <b>(b)</b> L-VDCC subunit expression was normalized to cardiac calsequestrin protein expression in the same sample (number of NF/F specimens was always identical for each subunit; n = 5–8). Quantitative analysis of subunit protein expression is depicted as ratio of F vs. NF. * p<0.001; ** p<0.0001. <b>(c)</b> mRNA expression of β-subunit isoforms (NF: n = 5; F: n = 9–13) was measured by real time PCR, and always normalized to cardiac calsequestrin mRNA expression. * p<0.05.</p

Protein expression of cardiac L-VDCC subunits in old wild-type and tg Ca_V1.2 mice

<div><p><b>(a)</b> Specimens from old wild-type mice and tg Ca_V1.2 in heart failure were analyzed in immunoblots using specific polyclonal antibodies directed against the particular L-VDCC subunits.</p><p><b>(b)</b> Protein expression of L-VDCC subunits was always normalized to cardiac calsequestrin protein expression in the same sample. Quantitative analysis of subunit protein expression is depicted as ratio of 10 months old tg Ca_V1.2 vs. age-matched wild-type. β₁ protein bands were faint, and thus not analyzed quantitatively (number of WT/old tg Ca_V1.2 specimens was always identical for each subunit; n = 4). * p<0.05.</p></div

Single L-VDCC gating of young and old tg Ca_V1.2 resembles data obtained from human non-failing and failing ventricle

<p>In a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone.0000292-Schroder1" target="_blank">[6]</a> we found single-channel activity to be significantly increased in ventricular myocytes from human hearts failing due to idiopathic dilated cardiomyopathy compared to non-failing ventricles. In excellent agreement the present study reveals activity of single L-VDCC from ≥9 months old, i.e. failing murine hearts overexpressing the human Ca_V1.2 to be significantly increased compared to single-channel activity in 4 months old, <b><i>i.e.</i></b> non-failing young transgenics obtained in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone.0000292-Groner1" target="_blank">[16]</a>. Charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV. Note that Schroder et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone.0000292-Schroder1" target="_blank">[6]</a> did not use a depolarizing bath solution, thus potentials are approximate values.</p>*<p>p<0.05 and ⁽*⁾p = 0.07 in a Student's t-test; ^†p<0.05 in a Mann-Whitney test (performed when data failed normality test). Numbers of experiments given in parentheses indicate number of experiments with only one channel in the patch.</p

tg mouse model with an inducible cardiac overexpression of the β_2a under control of a hybrid bombyx-ecdysone receptor

<div><p><b>(a)</b> For cardiac-specific expression the hybrid bombyx-ecdysone receptor (VgBmEcR) was placed under the control of αMHC promoter (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#s4" target="_blank">Materials and Methods</a>). Transgenic mice (tg_ind β_2a) positive for the hybrid bombyx-ecdysone receptor and the construct of the ecdysone response element (EcRE) and the β_2a, respectively, are identified in Southern blots. The radiolabeled probe specific for the coding sequence of VgBmEcR was generated by SacI digestion. It hybridized to a 3.7 kb band in transgene mouse genomic DNA digested by EcoRI digest; the radiolabeled DNA probe specific for the coding sequence of β_2a was generated by HindIII/KpnI digest. It hybridized to a 2.4 kb band of genomic DNA digested with HindIII/BamHI in tg_ind β_2a but not in WT. </p><p><b>(b)</b> 48 h after treatment with the inducing drug tebufenozide (+T) Western-blot analysis with ventricular tissue from 4–5 month old mice reveals increased expression of β₂-protein in tg_ind β_2a compared to treated wild-type or sham-induced transgenics.</p><p><b>(c)</b> Exemplary traces of single-channel recordings from murine ventricular myocytes. Induction of cardiac overexpression of the β_2a (+T) does not alter single-channel behavior compared to either wild-type mice after treatment with the inducing drug or i.p-application of only the vehicle (water/oil-emulsion) to β_2a-transgenic mice (“sham”). Data were obtained by patch-clamp recordings using cell-attached configuration (charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV for 150 ms). Bottom traces show ensemble average currents from the respective experiment.</p></div

Gating of single L-VDCC in ventricular myocytes from mice showing cardiac overexpression of Ca²⁺-channel subunits

<div><p><b>(a–e)</b> Single-channel gating parameters of ventricular L-VDCC from murine hearts. Compared to 4–5 months old mice showing a cardiac overexpression of the human Ca_V1.2 (tg Ca_V1.2), the inducing compound tebufenozide (T) significantly increased single L-VDCC activity in ventricular myocytes from age-matched double-transgenics (tg Ca_v1.2×tg_ind β_2a, showing an additional inducible β_2a-overexpression) 48 h after drug administration. Overexpression of the β_2a-subunit without overexpression of the human Ca_v1.2 does not alter single-channel gating (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone-0000292-g004" target="_blank">Figure 4c</a>). Tebufenozide treatment does not affect single-channel gating in ventricular myocytes from wild-type mice. Data were obtained by patch-clamp recordings using cell-attached configuration (charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV for 150 ms). *p<0.05 and ⁽*⁾p<0.09 compared to tg Ca_v1.2 in Student's t-test. Number of underlying experiments is given in parentheses.</p><p><b>(f)</b> Exemplary traces of single-channel recordings from murine ventricular myocytes. Activity of single L-VDCC is clearly higher in old (≥9 months, failing) tg Ca_V1.2 compared to channels from young (4–5 months, non-failing) tg Ca_V1.2. Induction of β₂-overexpression in hearts of young tg_ind β_2a×tg Ca_V1.2 by tebufenozide mimicks the heart-failure phenotype of L-VDCC gating otherwise not observed until tg Ca_V1.2 enter the “Maladaptive State” at an age ≥9 months. T = tebufenozide. Data were obtained by patch-clamp recordings using the cell-attached configuration (charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV for 150 ms). Bottom traces show average currents from the respective experiment.</p></div