Figure 6

<p>Position-effect blocker activity of the XB fragment. (A) Constructs introduced into the NIH3T3 cell line. Construct I (top) contains a CAGGS promoter-driven fluorescent protein (Venus) and a neomycin-resistance gene, enabling G418 selection of stable transformant colonies of transfected NIH3T3 cells. Construct II (bottom) is organized similarly, except it contains XB blocker fragments bordering both sides of the Venus expression marker gene. (B) Experimental scheme illustrating the transfection scenario. Stable transformants of both constructs were selected using G418. Colonies were maintained in culture for one year. We selected samples from each colony after 1, 6, and 12 months for FACS analyses. (C) Fluorescence assessment of 12 colonies harboring either Construct I or Construct II. Fewer fluorescent cells were observed in colonies lacking the XB fragment, while numerous fluorescent cells were observed in colonies harboring the XB fragment, indicating that the XB construct (Construct II) maintained Venus expression.</p

Figure 4

<p>Methylation profile of insulator fragment. (A) Hindbrain and forelimbs were dissected from 11 dpc embryos to obtain the DNA used for the methylation assays. (B) Map of the tested fragment. Red line represents the insulator (XB fragment) and blue line represents the regulator (BB fragment). Bisulfite-treated genomic DNA was subjected to PCR using three sets of primers (indicated by arrows; see Experimental Procedures). Primer pairs for one PCR reaction have matching arrow color. (C) Ten clones from each PCR product were sequenced. Their methylation status is shown here. White circles represent methylated cytosine residues, while black circles represent non-methylated cytosine residues.</p

Figure 3

<p>Insulation activity is spatially dependent. (A) Design of two targeted transgenic mice. XNs and XB as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000175#pone-0000175-g002" target="_blank">Figure 2A</a>. (B) Expression pattern of XNs- and XB-transgenic mouse embryos. XNs 11 dpc embryos expressed lacZ in the limbs and genital bud, whereas XB embryos did not. Limbs are shown in the boxed areas and genital buds are shown in the middle panels. (C) Scheme illustrating the regulation underlying enhancer-promoter interaction. Within the CNS, the XB fragment prevents interaction between the enhancer and <i>Hoxd13</i> promoter, while the BB fragment blocks the insulator activity of the XB fragment within the limbs and genital bud.</p

Figure 5

<p>Premature expression of <i>Hoxd13</i> observed in XB-targeted transgenic mice. (A) <i>Hoxd13</i> expression in 7 dpc embryos was specifically upregulated. Sample RNA levels were normalized according to β-actin mRNA levels. W1–W3, samples from three litters arising from wild-type mice; B1–B6, samples from six litters arising from internally bred BB-transgenic mice; X1–X8, samples from eight litters arising from internally bred XB-transgenic mice. (B) Hypothetical scheme for premature expression of these genes in XB-transgenic mice. The region downstream of <i>Evx2</i> recruits repression over the <i>Hox</i> complex before the 7-dpc embryonic stage in preparation for <i>Hox</i> expression in wild-type and BB-targeted transgenic mice. The XB fragment disrupts repression, preventing the repressor region downstream of <i>Evx2</i> from recruiting repression into the <i>HoxD</i> complex.</p

Ultrasound-induced cell killing in MES-SA and MES-SA/DX5 cells 24 hr post exposure to different acoustic intensities.

<p>(a) Cell viability assessed by WST-8 and cell counting assay. Asterisks (*) indicate the statistical significance of the difference between the absolute percentages obtained from WST-8 and cell counting assays at one intensity for one cell line. (b) Flow cytometric analyses for FITC-labelled Annexin V and PI staining. Vertically-aligned asterisks indicate the statistical significance of Annexin V (+)/PI (−) cells, whereas horizontally-aligned asterisks indicate the statistical significance of Annexin V (+)/PI (+) cells in comparison to control. Data points are presented as mean ± SEM.</p

Genes up- or down-regulated in HaCaT cells.

<p>The fold change is the ratio between control and each experiment area.</p

Effects of alkannin on IκB-α activity in UVB-exposed cells.

<p>(A) Cells exposed to alkannin or UVB light (40 mJ/cm²) alone or both. Western blot analysis was performed with IκB-α protein (30 µg protein in each group). (B) Immunofluorescent assay for the detection of NFκB p65 in: untreated cells (a), cells pre-treated with 1 µM alkannin for 24 h (b), cells exposed to UVB light (c), cells pre-treated with 1 µM alkannin for 24 h and then exposed to 40 mJ/cm² UVB light (d). Results are representative of five independent experiments (original magnification a–d: 200×). (C) NFκB from the nuclear extracts was analyzed by Western blotting, and histone H1 was used as a loading control for nuclear proteins. These data are representative of 3 independent experiments.</p

A summary of the differential responses of MES-SA and MES-SA/DX5 cells to US treatment.

(*)<p>denotes statistical significance between MES-SA and MES-SA/DX5 cells.</p

Verification of microarray results by qPCR assay.

<p>Cells exposed to alkannin or UVB light (40 mJ/cm²) or both were used for real-time qPCR assay of the selected genes BCOR and TBPL1. Each mRNA expression level was normalized with GADPH. Data are presented as mean ± SD (n = 3). *p<0.005, **p<0.05.</p

Dual treatment protocols.

<p>Cell survival following dual treatment protocols with doxorubicin (1 µM) and ultrasound (0.4 W/cm²). (a) Simultaneous treatment protocols. (b) Sequential treatment protocols. Data points are presented as mean ± SEM. Asterisks (*) denote the statistical significance of changes in cell survival following (Dox-US) protocol compared to (US 48) and (Dox 48) for each cell line. Following (US-Dox) treatment protocol, the cell survival of MES-SA cells decreased significantly compared to (US 48) but was insignificantly increased compared to (Dox 48). In MES-SA/DX5 cells, cell survival was unchanged. However, (US-Dox) protocol showed consistently large difference between the percentages obtained from WST-8 and cell counting assays indicating the presence of cells contributing to viability below the size threshold for detection by the cell counter.</p