環境管理計画への市民参加とその規定因としてのエンパワーメント

<p>DMPC/2/siRNA treatment of Jurkat T cells, 48 hours from the beginning of transfection.</p

Lyp protein expression in Jurkat T cells after siRNA s/a transfection using a commercial system.

<p>(A) siRNA transfection with siRNA having higher affinity to the target mRNA sequence (siRNA1) resulted in a reduction of Lyp O.D. (optical density) percentages to untreated cells to 44%, 43% and 48% with the doses of 40-60-80 pmols respectively after 48 hours of transfection. O.D. values of Lyp and β-actin were obtained with ImageLab software. Lyp O.D. values for every treatment were normalized to the corresponding β-actin values. No reduction was obtained with siRNA molecule with lower affinity (siRNA2). * indicates p<0.05. (B) Representative WB image with all the corresponding experimental groups is shown. Lyp O.D. % with respect to untreated cells’ O.D. values calculated as percentage to untreated cells’ O.D. ‘Untreated’ represents the basal control level (100%) of Lyp protein expression in cells cultured in RPMI. The efficacy of transfection is calculated from the difference between 100 and the test O.D. percentage in each experiment.</p

Circular dichroism spectroscopy.

<p>(A) CD spectra of 1.3 <b>μ</b>M siRNA in buffer solution (5 mM HEPES, 0.1 mM EDTA, pH 7.4). (B) CD spectra of 1.3 <b>μ</b>M siRNA in the DMPC/1 formulation. (C) CD spectra of 1.3 <b>μ</b>M siRNA in the DMPC/2 formulation. Spectra were recorded at 25°C.</p

Dynamic light scattering.

<p>Hydrodynamic diameter distribution functions (averaged by number) obtained by CONTIN analysis of DLS data (scattering angle 90°, T = 25°C) for DMPC/1 liposomes (panel A); DMPC/2 liposomes (panel B); DMPC/1/siRNA lipoplexes (panel C); DMPC/2/siRNA lipoplexes (panel D).</p

Confocal microscopy analysis of lipoplexes incorporation in Jurkat T cells.

<p>Data are shown after 60 minutes and 4 and half hours of incubation. Arrows indicate the localization of liposome fluorescence (red spots) compared to cell membrane (green) and nucleus (blue). Z-reconstructions obtained by confocal microscopy show the internalization of lipoplexes in Jurkat T cells in the X- and Y-axis projections. Plasma membrane is stained with wheat germ agglutinin (WGA), whereas nuclei are counterstained with Hoechst. Bar: 10 μm.</p

Evaluation of cell death after lipoplexes internalization.

<p>Flow cytometric analysis of HD PBMC (left) and Jurkat cells (right) treated with rhodamine-marked lipoplexes for 4 and a half hours. The histogram shows both the percentage of ‘transfected’ lymphocytes (rhodamine⁺ cells) and the percentage of dead cells among the transfected ones (DAPI⁺ and rhodamine ⁺ cells).</p

Lyp protein expression in Jurkat T cells 72 hours after the beginning of O/N transfection with different doses of siRNA s/a in DMPC/1/siRNA lipoplexes.

<p>Representative duplicate experiment among all replicas in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175784#pone.0175784.s004" target="_blank">S4 Fig</a>. (A) Lyp expression in Jurkat T cells after O/N transfection with DMPC/1 alone (DMPC/1), 20 pmols of siRNA alone (siRNA20) and 20 pmols of siRNA complexed with DMPC/1 (DMPC/1/siRNA20) or cultured in RPMI. 20 pmols of siRNA complexed with DMPC/1 resulted in a 15% reduction of Lyp expression. (B) Same experiment as in A using 60 pmols of siRNA (siRNA60) complexed with DMPC/1 (DMPC/1/siRNA60). 33% reduction of Lyp expression was obtained. (C) Same experiment as in A using 100 pmols of siRNA (siRNA100) complexed with DMPC/1 (DMPC/1/siRNA100). 45% reduction of Lyp expression was obtained. (D) Representative WB image within all experimental groups is shown.</p

Confocal microscopy analysis of Lyp protein expression in Jurkat T cells after transfection with lipoplexes.

<p>The comparison shown is between samples transfected with 60 and 100 pmols of Lipo/siRNA lipoplexes, and analyzed after 72 hours from the beginning of the O/N transfection. Control cells are untreated and cultured in RPMI (upper panels). Lyp protein expression is revealed by anti-mouse IgG conjugated to Alexa Fluor 555 (red signal). Cell morphology and DNA are detected by WGA (green) and Hoechst (blue) staining, respectively. Bar: 20 μm.</p

Lyp protein levels in Jurkat T cells 48 hours after the beginning of O/N transfection with different doses of siRNA s/a in DMPC/1/siRNA lipoplexes.

<p>Representative duplicate experiment among all replicas in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175784#pone.0175784.s004" target="_blank">S4 Fig</a>. (A) Lyp expression in Jurkat T cells cultured in RPMI or after O/N transfection with DMPC/1, 20 pmols of siRNA and 20 pmols of siRNA in DMPC/1/siRNA lipoplexes (DMPC/1/siRNA20). 20 pmols of siRNA complexed with DMPC/1 resulted in a 31% reduction of Lyp expression. (B) Same experiment as in A using 60 pmols of siRNA complexed with DMPC/1 (DMPC/1/siRNA60). 39% reduction of Lyp expression was obtained. (C) Same experiment as in A using 100 pmols of siRNA complexed with DMPC/1 lipoplexes (DMPC/1/siRNA100). 47% reduction of Lyp expression was obtained. (D) Representative WB image within all experimental groups is shown. Under each blot Lyp O.D. values for every treatment are normalized over the corresponding β-actin values. All percentages were expressed relatively to untransfected cells (RPMI) that is considered the 100% of basal Lyp expression. Graphs A, B, C show the mean values and their standard deviations. In all experimental conditions (<i>vide infra</i>), a decrease in Lyp protein level was not observed in cells treated with the liposome alone or, more importantly, in cells treated with the correspondent dose of the siRNA s/a free molecule.</p

Confocal microscopy analysis of Lyp protein expression in human PBMC after transfection with lipoplexes.

<p>The images show the concurrence of the lipoplexes (red dot/arrow) inside CD3⁺ (white) and CD3^- cells and the reduction in Lyp immunofluorescence signal (green). Cells nuclei are counterstained with Hoechst dye (blue) in comparison with untransfected cells. Bar: 20 μm.</p