Lentiviral transduction and establishment of transgenic hNPCs.

<p>(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. <i>ns</i>, non-significant (p > 0.5). Scale bar, 20 μm.</p

The S26/28 phosphorylation site is not necessary for <i>bdv-p</i>-induced reduction in GABAergic neurogenesis.

<p>(A) Schematic representation of P and Paass. Paass is mutated in S26/28 residues and thus lacks the corresponding phosphorylation site. (B) hNPCs were transduced with lentiviral vectors bearing the <i>bdv-p</i> or <i>bdv-p</i>_<i>aass</i> gene and immunostained at the undifferentiated stage (day 0) with an antibody directed against the P protein (green). Nuclei were stained with DAPI (blue). Transduction efficiency was evaluated by enumeration of P-positive cells (right panel). (C) Western blot showing the level of P in <i>bdv-p</i> and <i>bdv-p</i>_<i>aass</i>-expressing hNPCs at 14 days of differentiation. P is normalized to actin. (D) Transgenic hNPCs and their NT matched controls were induced to differentiate for 14 days and GABAergic neurons were quantified. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p = 0.002. Scale bar, 20 μm.</p

<i>bdv-p</i> alters the expression of <i>ApoE</i> and <i>Noggin</i>.

<p><i>bdv-p</i>- and <i>bdv-x</i>-expressing-hNPCs and their matched NT controls were induced to differentiate for 0, 4 or 14 days before RNA and protein analyses. <i>ApoE</i> expression was measured by RT-qPCR at (A) day 4, (B) day 0 and (C) day 14. (D) Western blot analysis showing ApoE level. It was normalized to actin. <i>Noggin</i> expression was measured by RT-qPCR at (E) day 4, (F) day 0 and (G) day 14. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001.</p

Expression of the <i>bdv-p</i> but not <i>bdv-x</i> gene in hNPCs impairs neuronal differentiation.

<p>Transduced hNPCs expressing <i>gfp</i>, <i>bdv-p</i> or <i>bdv-x</i> genes and their matched NT controls were induced to differentiate for 14 days. (A) Immunostaining with antibodies directed against βIII-Tubulin, a neuronal marker (red), or GFAP, an astrocytic marker (green). Nuclei were stained with DAPI (blue). For panel homogenization, GFAP immunostaining performed in <i>gfp</i>-expressing hNPCs was re-colored in green. The percentage of neurons and astrocytes was determined based on enumeration of βIII-Tubulin-, GFAP- and DAPI-positive cells in (B) <i>gfp</i>-expressing hNPCs, (C) <i>bdv-p</i>-expressing hNPCs and (D) <i>bdv-x</i>-expressing hNPCs. Results in B, C and D are representative of 2, 5 and 2 independent experiments, respectively. All experiments were performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001, ns, non-significant (p > 0.5). N, neurons. As, astrocytes. Scale bar, 50 μm.</p

<i>bdv-p</i> alters the expression of <i>TH</i> and <i>Scg10/Stathmin2</i>.

<p><i>bdv-p</i>- and <i>bdv-x</i>-expressing- hNPCs and their matched NT controls were induced to differentiate for 0, 4 or 14 days before RNA and protein analyses. <i>TH</i> expression was measured by RT-qPCR at (A) day 4, (B) day 0 and (C) day 14. (D) Western blot analysis showing TH level. <i>Scg10/Stathmin2</i> expression was measured by RT-qPCR at (E) day 4, (F) day 0 and (G) day 14. (H) Western blot analysis showing SCG10/Stathmin2 level. TH and SCG10/Stathmin2 were normalized to actin. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001.</p

Expression of <i>bdv-p</i> or <i>bdv-x</i> gene does not alter hNPCs at the undifferentiated stage.

<p>RNAs from <i>bdv-p-</i> and <i>bdv-x</i>-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of <i>bdv-p</i> and <i>bdv-x</i>-expressing hNPCs was analyzed by BrdU labeling (C) and by a mitochondrial dehydrogenase activity-based assay (D and E) in the presence of growth factors and by enumeration of DAPI-positive cells (F and G) in the absence of growth factors. Results in A and B are representative of two independent experiments performed in triplicate. Results in C represent the mean of two independent experiments performed in quintuplicate. Results in D and E are representative of 2 independent experiments performed in quintuplicate. Results in F and G are from 1 experiment performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. <i>ns</i>, non-significant (p > 0.5).</p

<i>bdv-p</i> alters the molecular program involved in neurogenesis.

<p><i>bdv-p</i>-expressing-hNPCs and their matched NT controls were induced to differentiate for 4 days and RNA was analyzed using an RT-PCR array. The 84 human genes analyzed are shown. A color code shows the differential expression of the genes, from green (non-regulated) to red (the most regulated). Genes were grouped depending on their known function in neurogenesis. Up to the dark line are the genes significantly modified after application of an arbitrary cut-off of 3. -, genes down-regulated. +, genes up-regulated.</p