Metallic Nanoshells with Semiconductor Cores: Optical Characteristics Modified by Core Medium Properties

It is well-known that the geometry of a nanoshell controls the resonance frequencies of its plasmon modes; however, the properties of the core material also strongly influence its optical properties. Here we report the synthesis of Au nanoshells with semiconductor cores of cuprous oxide and examine their optical characteristics. This material system allows us to systematically examine the role of core material on nanoshell optical properties, comparing Cu2O core nanoshells (εc ∼ 7) to lower core dielectric constant SiO2 core nanoshells (εc = 2) and higher dielectric constant mixed valency iron oxide nanoshells (εc = 12). Increasing the core dielectric constant increases nanoparticle absorption efficiency, reduces plasmon line width, and modifies plasmon energies. Modifying the core medium provides an additional means of tailoring both the near- and far-field optical properties in this unique nanoparticle system

Recruitment of NURF and dREAM is dependent on dCTCF and CP190 at specific sites.

<p>ChIP in S2 cells treated with dsRNA against dCTCF and CP190 (dsCTCF/dsCP190; dark colors) or against luciferase as control (dsLuci; light colors). Antibodies were used specific for dCTCF, CP190 and components of the NURF (ISWI, Chro) and dREAM complex (Mip40, Mip120, Mip130) or, as part of both complexes, CAF-1/p55. Error bars indicate the standard deviation of three independent experiments. (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).</p

NURF binding causes nucleosomal depletion at CP190 binding sites.

<p>(<b>A</b>) Cumulative representation of changes in H3-binding and MNase-protection as detected by H3 ChIP-seq and MNase-seq after depletion of CTCF/CP190 (green) or ISWI (orange). Data is shown as coverage for specific knock-down normalized to luciferase control knock-down (luci) after log2-transformation. Average effects are shown across CP190 binding sites (colored) or control sites shifted 25 kb (grey). (<b>B</b>) All sites with increased H3 binding after CTCF/CP190 depletion (responders) show a similar H3 increase upon ISWI depletion. Non-responding sites after CTCF/CP190 depletion (non-responders) do not respond to ISWI depletion. H3 ChIP in S2 cells treated with dsRNA against CTCF and CP190 (dsCTCF/CP190; green), ISWI (dsISWI; orange) or against luciferase as control (dsLuci; black). Error bars indicate the standard deviation of two independent experiments (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).</p

NURF and dREAM components co-localize with CP190 genome-wide.

<p>(<b>A</b>) Correlation analysis for genome-wide binding of CP190 with 215 profiles from S2 cells (modENCODE) and 5 profiles from Kc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765-Georlette1" target="_blank">[33]</a>. Shown are the top 30 ranking factors. Components of the NURF complex are marked in pink and of the dREAM complex in blue. (<b>B</b>) Cluster heat map of 6,000 genomic regions with CP190 and/or CTCF sites compared with binding sites for components of NURF (NURF301, ISWI, Chro) and dREAM (E2F2, Lin-52, Mip120, Mip130, Myb) complexes. Each lane represents an 8 kb region. Scale represents binding (red) to no binding (blue).</p

Insulator specific effects of NURF and dREAM components.

<p>S2 cells pools with the integrated luciferase reporter constructs with different CTCF/CP190 binding sites located between the enhancer (O, OpIE2) and the promoter of the reporter gene (L, <i>luciferase</i>). (<b>A</b>) Luciferase activity after control knockdown of GFP shows the enhancer blocking activity of <i>Fab-8</i> (F8), bicoid (bcd), CG31472 and <i>Fab-6</i> (F6(2)), when compared to the CTCF binding site mutant (F8mut) (top). Error bars indicate the standard deviation of three biological replicates. The different insulator reporter constructs are depicted (bottom), the genomic fragments used are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765.s013" target="_blank">Table S2</a> and genome browser views are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765.s007" target="_blank">Figure S7</a>. (<b>B</b>) Knockdown experiments against CTCF, ISWI or NURF301 (top) and of CTCF, CAF1/p55 or triple-knockdown of Mip-factors (bottom). Fold change of luciferase activity is calculated relative to the control knockdown. Error bars indicate the standard error of three or more individual replicates (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).</p

RNA interference (RNAi) identifies 78 factors inducing insulator reporter gene activity including NURF and dREAM components.

<p>(<b>A</b>) Workflow of the RNAi screen in 66×384-well plates from the DRSC. Knockdown of 13900 genes was done with <i>Drosophila</i> S2 cells with the integrated F8OF8L insulator reporter construct (F8, <i>Fab-8</i>; O, OpIE2 enhancer; L, <i>luciferase</i>). (<b>B</b>) Top GO-terms (determined via GeneCodis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765-CarmonaSaez1" target="_blank">[58]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765-TabasMadrid1" target="_blank">[60]</a>) for the 78 identified genes. (<b>C</b>) High-throughput data shown in a dotplot diagram. Z-scores are indicated for every well (well number). For many gene products several wells contain different dsRNA sequences targeting the same gene. Z-scores higher than two are highlighted in red. (<b>D</b>) Individual depletion of NURF and dREAM components and associated factors verify enhancer blocking function. S2 cell pools with the integrated F8OF8L insulator reporter (dark grey) or the control F8OL reporter construct (light grey) were incubated with dsRNA against factors of the NURF-complex (pink): ISWI, NURF-38, CAF1/p55, NURF301, Pzg, DREF or against the dREAM-complex (blue): CAF1/p55, Mip40, Mip130, E2F2. Reporter gene activity is expressed as fold change relative to control knockdown. Error bars indicate the standard deviation of three individual replicates. (p-values: *≤0.05, **≤0.01, ***≤0.001).</p

NURF and dREAM components co-localize with dCTCF/CP190.

<p>ChIP in S2 cells with antibodies against CTCF and CP190 and components of the NURF complex (ISWI, NURF301, Pzg and Chro) and dREAM complex (Mip40, Mip120, Mip130, E2F2) or CAF-1/p55. The genomic regions tested are indicated (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765.s013" target="_blank">Table S2</a>) and grouped into CTCF plus CP190, low CTCF plus CP190, low CTCF without CP190 and neither CTCF nor CP190. Error bars indicate the standard deviation of three independent experiments.</p

Purification of either CTCF or CP190 reveals NURF and dREAM binding to both insulator factors.

<p>(<b>A</b>) Interaction heatmap based on Mascot scores (dCTCF) or fold enrichment of normalized intensities (CP190), depicting associated factors identified by mass spectrometry after immunopurification of FLAG-dCTCF or FLAG-CP190 expressed in S2 cells. (<b>B</b>) Nuclear extracts from S2 cells (lanes 1–2, 7–10) and S2 cells stably expressing FLAG-CP190 (lanes 3–4) or FLAG-dCTCF (lanes 5–6) were precipitated with FLAG antibody (lanes 2, 4, 6), CP190 antibody (lane 9), dCTCF antibody (lane 10) or IgG (lane 8) as control. Antibodies used in Western blot are indicated on the right. Lanes 1, 3, 5 and 7: 1% Input.</p

RT-qPCR-based confirmation of RNA-sequencing results using both LNA GapmeRs against TUG1.

HUVECs were transfected with two LNA GapmeRs against TUG1—LNA TUG1_1 and LNA TUG1_2 –and LNA Ctrl and expression levels of (A) VAMP4, (B) TOR1AIP2, (C) KAT6B and (D) ABCA1 were measured after 48 hours by RT-qPCR. Expression is relative to P0 (n = 4; SEM; RM one-way ANOVA with Greenhouse-Geisser correction and Holm-Sidak multiple comparison test). (TIF)</p

<i>TUG1</i> is not important for basal cell turnover, barrier or mitochondrial function, migration and monocyte adhesion.

(A)–(G) HUVECs were transfected with two LNA GapmeRs against TUG1—LNA TUG1_1 and LNA TUG1_2 –and LNA Ctrl (10 nM) and (A) expression levels were measured after 48 hours by RT-qPCR. Expression is relative to GAPDH (n = 4; SEM; RM one-way ANOVA with Greenhouse-Geisser correction and Sidak multiple comparison test). (B) Relative cell growth determined from cell count at 0 h, 24 h, 48 h and 72 h (n = 3; SEM; RM Two-way ANOVA with Tuckey multiple comparison test). (C) Caspase-3/7 activity was measured by determination of fluorescence with ELISA plate reader (n = 3; SEM; One-way ANOVA with Holm-Sidak correction). Staurosporine was taken along as a postive control. (D) Cell-cell interactions (Rb) and cell-matrix-interactions (α) were measured by Electric Cell Impedance Sensing (ECIS; n = 3; SEM; Kruskal-Wallis-test with Dunn´s correction). (E) Determination of re-establishment of monolayer after wounding using ECIS (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test). (F) Seahorse mitochondrial stress test assessing multiple mitochondrial characteristics via measurement of changes in Oxygen Consumption Rate (OCR) after serial injection of Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone A/Antimycin (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test. One representative experiment displaying the changes of OCR throughout the progress of the Seahorse mitochondrial stress test assay. (G) Assessment of monocyte adhesion with and without TNF-α stimulation. (n = 3; SEM; Two-way ANOVA with Tuckey multiple comparison test).</p