Analyses of methylation status of CGIs associated <i>mir-142</i> and <i>in vitro</i> methylation of its upstream regulatory region.

<p>(<b>A</b>) Methylation-specific PCR (MSP) analyses of the CGIs, before and after treatment with 5-Aza-2′-deoxycytidine (5-Aza). U and M, unmethylated and methylated products. Both standard gel images and 3D densitograms of the signal intensities are shown. (<b>B</b>) Schematic representation of the <i>mir-142</i> locus. Locations of MSP amplicon #1 and #2 are indicated by arrows. 18 CpGs (region #1) and 14 CpGs (region #2) were subjected to bisulfite sequencing. CpG-containing transcription factor binding motifs are enclosed by boxes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#pone-0079231-g003" target="_blank">Figure 3B</a> for more information). E-box, enhancer box. Numbers indicate the position relative to <i>pre-miR-142</i> (+1 to +87). (<b>C</b>) Bisulfite sequencing of DNA from MG-63 cells before and after treatment with 5-Aza for 72 hours, and untreated IOR/OS14 cells. The Wilcoxon signed rank test was used to test for statistical differences between treated and untreated MG-63 cells, as described in more details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#s2" target="_blank">Materials and Methods</a>. A <i>P</i> value ≤0.05 was considered as significant. (<b>D</b>) Bisulfite sequencing of immortalized bone marrow-derived stromal cells (iMSC#3) and primary osteoblasts. (<b>E</b>) Bisulfite sequencing of K562 leukemia- and peripheral blood progenitor cells. Black and white circles represent methylated and unmethylated CpGs, respectively, and each row represents a single clone. Grey circles, not determined. Ten clones were sequenced (n = 10), with the exception of MG-63 cells (region #1, n = 13; region #2, n = 12). (<b>F</b>) The 2,031 bp upstream region of <i>pre-mir-142</i> was cloned into the promoter-less luciferase reporter construct pCpGL-basic and <i>in vitro</i> methylated with M.SssI (pCpGL/2031_M.SssI) or mock-methylated (pCpGL/2031_mock). All three constructs were individually transfected into U-2 OS cells along with a <i>Renilla</i> reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences and the <i>P</i> values are shown above the histograms. A <i>P</i> value ≤0.05 was considered as significant.