Published studies on methylation of mammalian mtDNA determined using bisulfite pyrosequencing or deep sequencing.

<p>Published studies on methylation of mammalian mtDNA determined using bisulfite pyrosequencing or deep sequencing.</p

Enrichment of human and mouse mtDNA over nuclear DNA.

<p>Ratios of copy numbers of human (A) or mouse (B) mtDNA and nuclear DNA were determined by qPCR of mtDNA and nuclear DNA marker genes (Mean±SD, triplicated assays). Numbers show fold enrichment compared to total DNA.</p

Shotgun bisulfite sequencing of human iPSC mtDNA.

<p>(A) Ratios of copy numbers of human mtDNA and nuclear DNA were determined by qPCR of mtDNA and nuclear DNA marker genes (Mean±SD, triplicated assays). Numbers show fold enrichment compared to total DNA. (B) CpG methylation of mtDNA-encoded genes (box plot; boxes and whiskers indicate quartiles and minimum/maximum values, horizontal lines in boxes indicate median). Numbers at the top indicate <i>p</i>-values of 2-tailed t-test against unmethylated lambda DNA. (C, D) Cytosine methylation of mtDNA (C) and unmethylated lambda DNA (D). Percentage of cytosine methylation in the CpG and non-CpG contexts is shown with red and blue dots, respectively. Deep sequencing read coverage at cytosines is shown with green dots. In panel (C), locations of mtDNA-encoded genes and D-loop are indicated at the top, where positions of tRNA genes are shown with vertical bars without gene names.</p

Targeted bisulfite deep sequencing of purified, linearized human mtDNA using hND1 primer.

<p>(A) Methylation of nine CpG sites in the mitochondrial ND1 gene determined based on the C/T SNP ratio. CpG sites #1 - #5 (shown in bold) correspond to the same names of CpG sites shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192722#pone.0192722.g004" target="_blank">Fig 4</a>. (B) Deep sequencing coverage tracks showing C/T SNPs at CpG sites #0 - #8 indicated in panel A. CpG and non-CpG cytosines are indicated by red and blue arrows, respectively. Pileup coverages of C/T SNPs are indicated with brown C (unconverted) and green T (converted) bars, respectively.</p

False-positive detection of bisulfite-resistant cytosines (brCs) in the negative control amplicons (NCAs) of mtDNA by bisulfite pyrosequencing.

<p>BrCs were detected in gel-purified or unpurified, bisulfite-converted PCR amplicons by pyrosequencing for 15 CpG sites: 5 in human ND1, 5 in human CYTB, 2 in mouse ND1, and 3 in mouse CYTB. The <i>p</i>-value was calculated using paired t-test (two-tails). The lines show mean ± 95% Confidence Intervals.</p

Detection of bisulfite-resistant cytosines in purified, linearized human mtDNA by bisulfite pyrosequencing using converted template-selective (A9515) and unselective (hND1) sequencing primers.

<p>Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using <i>in vitro</i> partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).</p

CSK is required for fulvestrant-induced ERα protein degradation in MCF-7 cells.

<p>(A, B) RNAi knockdown of CSK protein expression caused resistance of intracellular ERα protein to fulvestrant-induced degradation: Western blotting. Cells were infected with control (pLKO.1) or two CSK-knockdown shRNA lentivirus clones and subjected to exposure to fulvestrant. Expression of ERα protein was determined by Western blotting at varying time points of exposure (A). Intensities of ERα protein bands were determined by densitometry (B, mean±SEM of three independent experiments. Asterisk indicates statistical significance, p<0.05). (C) Similar experiments as shown in panels (A, B) were performed, but amounts of ERα protein in total cellular protein were determined by ELISA (mean±SEM of three independent experiments; *, p<0.05 to vehicle control; #, p<0.05 to pLKO.1-infected cells exposed to fulvestrant for the same period).</p

RNAi knockdown of CSK does not affect MCF-7 cell sensitivity to tamoxifen or paclitaxel.

<p>Cells were infected with empty lentivirus vector (pLKO.1) or two independent clones of lentiviruses expressing different shRNA species targeting CSK shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060889#pone-0060889-g001" target="_blank">Figure 1</a> (CSK KD#1 and #2) and then exposed to 1 µM 4-hydroxytamoxifen (4-OHT) for 10 days (A) or 1–1000 nM paclitaxel for 2 days (B). Cell viability was determined by crystal violet staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060889#pone.0060889.s003" target="_blank">Fig. S3</a>) and quantified by spectrophotometry (mean±SEM of three or more independent experiments).</p

Both fulvestrant and 17β-estradiol (E2) enhance proteasomal degradation of ERα protein in MCF-7 cells.

<p>(A–C) Fulvestrant (A) and E2 (B) caused time-dependent reduction in ERα protein expression: Western blotting. Intensities of ERα protein bands were determined by densitometry (C, mean±SEM of three independent experiments. Asterisks indicate statistical significance, p<0.05 to vehicle control). (D, E) E2 dose-dependent reduction in ERα protein expression. Cells were exposed to varying concentrations of E2 for 6 hours and subjected to Western blotting analysis of ERα protein (D). Intensities of ERα protein bands were determined by densitometry (E, mean±SEM of three independent experiments. Asterisk indicates t-test significance p<0.05 to vehicle control). (F–H), Pre-exposure to MG132 dose-dependently prevented reduction in ERα protein expression caused by fulvestrant (F) and E2 (G). Con, vehicle control (0.1% ethanol). Cells were exposed to varying concentrations of MG132 for 30 minutes and then exposed additionally to fulvestrant or E2 for 6 hours. Intensities of ERα protein bands were determined by densitometry (H, mean±SEM of three independent experiments. Asterisks indicate statistical significance, p<0.05).</p

Prenatal Exposure to BPA Alters the Epigenome of the Rat Mammary Gland and Increases the Propensity to Neoplastic Development

<div><p>Exposure to environmental estrogens (xenoestrogens) may play a causal role in the increased breast cancer incidence which has been observed in Europe and the US over the last 50 years. The xenoestrogen bisphenol A (BPA) leaches from plastic food/beverage containers and dental materials. Fetal exposure to BPA induces preneoplastic and neoplastic lesions in the adult rat mammary gland. Previous results suggest that BPA acts through the estrogen receptors which are detected exclusively in the mesenchyme during the exposure period by directly altering gene expression, leading to alterations of the reciprocal interactions between mesenchyme and epithelium. This initiates a long sequence of altered morphogenetic events leading to neoplastic transformation. Additionally, BPA induces epigenetic changes in some tissues. To explore this mechanism in the mammary gland, Wistar-Furth rats were exposed subcutaneously via osmotic pumps to vehicle or 250 µg BPA/kg BW/day, a dose that induced ductal carcinomas <i>in situ</i>. Females exposed from gestational day 9 to postnatal day (PND) 1 were sacrificed at PND4, PND21 and at first estrus after PND50. Genomic DNA (gDNA) was isolated from the mammary tissue and immuno-precipitated using anti-5-methylcytosine antibodies. Detection and quantification of gDNA methylation status using the Nimblegen ChIP array revealed 7412 differentially methylated gDNA segments (out of 58207 segments), with the majority of changes occurring at PND21. Transcriptomal analysis revealed that the majority of gene expression differences between BPA- and vehicle-treated animals were observed later (PND50). BPA exposure resulted in higher levels of pro-activation histone H3K4 trimethylation at the transcriptional initiation site of the alpha-lactalbumin gene at PND4, concomitantly enhancing mRNA expression of this gene. These results show that fetal BPA exposure triggers changes in the postnatal and adult mammary gland epigenome and alters gene expression patterns. These events may contribute to the development of pre-neoplastic and neoplastic lesions that manifest during adulthood.</p></div