Increased numbers of cells positive for NeuroD1 and NeuN in nodules of MB allograft treated with AAV-<i>Tis21</i> viral particles.

<p>A) Representative images of IHC staining for the differentiation marker NeuroD1 (red) and for the nuclei dye Hoechst (blue). Scale bars: 50 μm. B) Quantification of the percentage ratio of cells positive for NeuroD1 in nodules of MB allograft treated with AAV-<i>Tis21</i> (orange) and AAV-CBA (blue), to the total number of cells (Hoechst-positive). **** p < 0.0001, Mann-Whitney U test. Mice analyzed: n = 7 for AAV-CBA, n = 7 for AAV-<i>Tis21</i>; fields analyzed: n = 201 for AAV-CBA, n = 207 for AAV-<i>Tis21</i>. C) Representative images of IHC staining for the differentiation marker NeuN (green). In blue the nuclei. Scale bars: 50 μm. D) Quantification of the percentage of NeuN positive cells to the total number of cells (Hoechst-positive). Orange bar (AAV-<i>Tis21</i>), blue bar (AAV-CBA). **** p < 0.0001, Mann-Whitney U test. Mice analyzed: n = 2 for AAV-CBA, n = 3 for AAV-<i>Tis21</i>; fields analyzed: n = 31 for AAV-CBA, n = 41 for AAV-<i>Tis21</i>.</p

Inhibition of <i>Ptch1</i>^+/- MB growth in an allograft murine model by <i>Tis21</i> overexpression.

<p>A) Tumor volumes of nodules obtained by grafting primary <i>Ptch1</i>^+/- MB tumor cells subcutaneously in nude mice flanks. The bars indicate the average ± SEM of the tumor volume at the time of the viral particles injection (T1-T6) and 24 hours after the last treatment (pT6) with PBS (green), AAV-<i>Tis21</i> (orange) and AAV-CBA (blue). Groups of 3–5 mice treated with AAV-<i>Tis21</i> and AAV-CBA in three independent experiments and groups of 2 mice treated with PBS in two independent experiments were analyzed. A two-way ANOVA followed by post-hoc Bonferroni's multiple comparison test was used for the statistical analysis; ** p < 0.01, *** p < 0.001, **** p < 0.0001. B) Representative images of AAV-CBA, PBS and AAV-<i>Tis21</i>-treated nodules are shown. Scale bars: 10 mm.</p

AAV-<i>Tis21</i> infects DAOY and D283 MB cell lines and decreases <i>Cyclin D1</i> mRNA expression levels in <i>Tis21</i> dose-dependent manner.

<p>Real-time PCR analysis of mRNA obtained from DAOY (A and B) and D283 (C and D) cells infected with AAV overexpressing <i>Tis21</i> (AAV-<i>Tis21</i>) or AAV-CBA (control). Increasing MOI employed for infection is indicated in graphs. Average ± SEM values are from at least three independent qPCR experiments and are shown as fold change relative to the control sample (cells infected with the MOI 0.13x106 of AAV-CBA), which was set to unit. Human <i>TATA-binding protein</i> mRNA was used as endogenous control for normalization. Two-way ANOVA followed by Fisher's PLSD test was used for the statistical analysis; * p < 0.05, ** p < 0.01, or **** p < 0.0001, n.s. p non-significant, PLSD ANOVA test.</p

Primer list for qRT-PCR.

<p>Primer list for qRT-PCR.</p

Decreased numbers of cells positive for Ki67 and BrdU in nodules of MB allograft treated with AAV-<i>Tis21</i> viral particles.

<p>A) Representative confocal images of IHC staining for the proliferation marker Ki67 (green) and for the nuclei dye Hoechst (blue). Scale bars: 50 μm. B) Quantification of the percentage ratio of cells positive for the proliferation marker Ki67 to the total number of cells (Hoechst-positive) in nodules of MB allograft treated with AAV-<i>Tis21</i> (orange) and AAV-CBA (blue). **** p < 0.0001, Mann-Whitney U test; mice analyzed: n = 7 for AAV-CBA, n = 8 for AAV-<i>Tis21</i>; fields analyzed: n = 207 for AAV-CBA, n = 241 for AAV-<i>Tis21</i>. C) Representative confocal images of cells that have entered the cell cycle S-phase, either of AAV-<i>Tis21</i>-treated or of AAV-CBA-treated MB allograft, identified as BrdU+ cells (green). Sections are counterstained with Hoechst 33258 to visualize the nuclei. Scale bars: 50 μm. D) Quantification of proliferating cells, measured as mean ± SEM percentage ratio between number of BrdU+ cells and total number of cells (Hoechst-positive) in nodules of MB allograft treated with AAV-<i>Tis21</i> (orange) and AAV-CBA (blue). **** p < 0.0001, Mann-Whitney U test; mice analyzed: n = 4 for AAV-CBA, n = 4 for AAV-<i>Tis21</i>; fields analyzed: n = 144 for AAV-CBA, n = 144 for AAV-<i>Tis21</i>.</p

Carbon Nanotubes Promote Growth and Spontaneous Electrical Activity in Cultured Cardiac Myocytes

Nanoscale manipulations of the extracellular microenvironment are increasingly attracting attention in tissue engineering. Here, combining microscopy, biological, and single-cell electrophysiological methodologies, we demonstrate that neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes interact with carbon nanotubes by forming tight contacts and show increased viability and proliferation. Furthermore, we observed changes in the electrophysiological properties of cardiomyocytes, suggesting that carbon nanotubes are able to promote cardiomyocyte maturation

Additional file 1 of EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening

Supplementary Material 1: Table S1. Composition of the pilot AAV9 poo