Dependence of dentate gyrus growth on BRaf.

<p>(A) Generation of conditional <i>BRaf</i> mice. In the conditional <i>BRaf</i> allele exon 3 encoding part of the Ras-binding domain is flanked by <i>loxP</i> sites (arrowheads). Deletion of the neomycin resistance gene in <i>BRaf ^nfl</i> mice generated the <i>BRaf ^fl</i> allele. Deletion of both exon 3 and the neomycin resistance gene generated the <i>BRraf ^del</i> allele. Positions of primers used in PCR reactions to distinguish the different alleles are shown, for details see text. (B) Analysis of BRaf expression in embryos. Western blot analysis of BRaf expression in E10.5 embryos resulting from <i>BRaf ^wt/del</i> intercrossing reacted with antibodies against BRaf N-terminal or C-terminal epitopes. Note that the C-terminal-specific antibody detects a ∼82 kDa BRaf band in extracts from <i>BRaf ^del/del</i> and <i>BRaf ^wt/del</i> embryos that is smaller than the BRaf doublet bands of ∼92 and ∼89 kDa seen in wild-type embryos. Detection of β-actin served as loading control. (C) Analysis of downstream targets of BRaf signalling. The phosphorylation levels of the kinases ERK1,2, as well as the levels of the early growth response 1 transcription factor Egr1 were significantly reduced in the hippocampus of cKO mice compared to ctrl mice whereas the expression of Erk1,2 was unaltered. The residual level of BRaf in cKO may occur from “escaper” cells. Gapdh served as loading control. (D) Analysis of BRaf expression by immunohistochemistry. Upper panels are representative sagittal sections of P21 hippocampus immunostained for BRaf with an antibody against the BRaf N-terminus. Lower panels are images taken from boxed regions in upper panels; note presence of BRaf stain in cell body of singular granule neurons (arrows) and their dendrite extending into the molecular layer that might have “escaped” Cre recombinase-mediated <i>BRaf</i> deletion in <i>Nestin-Cre/BRaf ^fl,fl</i> mice. Scale bars; upper row, 200 µm; lower row, 25 µm. (E) Representative sagittal sections of P12 and P21 hippocampus stained with Nissl. Scale bars; 800 µm. (F) Volume of hippocampal granule cell layer (gcl) in 12 and 21 day old mice. Data are mean ±s.e.m.; P12, n = 3; P21, n = 7. (G) Exon organization and location of regulatory regions in BRaf isoforms. Boxes indicate exons with their sizes in nucleotides aligned to the regulatory, catalytic and RAS-binding domains (RBD) of BRaf protein. The vertical arrows above exon 3 indicate the positions of the 5′ end and 3′ end, respectively of an intron that has been spliced out in the small cDNA harbouring exon 3* in embryonic RNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058259#pone.0058259.s002" target="_blank">Figure S2</a>). This in-frame splicing retains the reading frame and is predicted to encode the 89 kDa BRaf isoform. The scheme is deduced from cDNA sequencing of wild-type and exon 2–4 spliced BRaf del samples (see text). The molecular masses of BRaf proteins present on the gel (Fig. 1B) are shown.</p

<i>Nestin-Cre</i> mediated deletion of <i>braf</i> impairs neuronal differentiation in the granular cell layer of the dentate gyrus.

<p>(A) Quantification of BrdU-labelled cells in the dentate gyrus of ctrl or cKO mice. Cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification 24 hours after the second BrdU pulse. Representative sagittal sections of the dentate gyrus stained with the proliferation marker BrdU (green). Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm. (B) Quantification of BrdU-labelled cells in the dentate gyrus at P22 of ctrl or cKO mice. Neural progenitor cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification of mice at P22. Representative sagittal sections of the dentate gyrus stained with proliferation marker BrdU (green). Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm. (C) Quantification of BrdU/NeuN-positive cells in the granular cell layer of the dentate gyrus of ctrl cKO mice. Neural progenitor cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification of mice at P22. Representative sagittal sections of the dentate gyrus stained with proliferation marker BrdU (green) and neuronal marker NeuN (red) 11–12 days after BrdU labelling. Double positive cells are marked with an arrow. Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm. (D) Quantification of BrdU/GFAP-positive radial glia cells in the granular cell layer of the dentate gyrus of ctrl or cKO mice. Neural progenitor cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification of mice at P22. Representative sagittal sections of the dentate gyrus stained with proliferation marker BrdU (green) and neural precursor/astrocyte marker GFAP (red) 11–12 days after BrdU labelling. Expanded region is indicated by an arrow; the arrowhead depicts a double-positive cell. Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm.</p

<i>Nestin-Cre</i> mediated deletion of <i>BRaf</i> impairs formation of synaptic networks of cultured hippocampal neurons.

<p>(A) Cells from the hippocampi of newborn mice were cultured for 6 days in vitro, fixed and stained for expression of BRaf and Map2. Scale bar = 25 µm<b>.</b> (B) Quantification of BRaf-positive, Map2-positive cells as a fraction of DAPI-labelled cells isolated from hippocampi at P0/P1 of ctrl or cKO mice and grown for 6 days in vitro. Data are mean ±s.e.m.; n = 5.</p

Cell cycle and cell fate analysis in postnatal hippocampus lacking BRaf.

<p>(A) Quantification of activated caspase-3-positive cells in the dentate gyrus at P24. Representative sagittal sections of the dentate gyrus of ctrl or cKO mice were stained for activated caspase-3 (brown); tissue was counterstained with Nissl. Data are mean ±s.e.m.; n = 7. Scale bar = 50 µm. (B) Quantification of BrdU-labelled cells in the dentate gyrus at P20 (2 h BrdU pulse) of ctrl or cKO mice. Data are mean ±s.e.m.; n = 4. (C) Quantification of Ki67-labelled cells in the dentate gyrus at P20 of ctrl or cKO mice. Data are mean ±s.e.m.; n = 4. (D) BrdU-positive Ki67-negative cells as a fraction of BrdU-labelled cells in the dentate gyrus at P20 of ctrl or cKO mice. Data are mean ±s.e.m.; n = 4. (E) Representative sagittal sections of the dentate gyrus of ctrl or cKO mice stained with the S-phase marker BrdU (green) and the proliferation marker Ki67 (red). Double positive cells are marked with an arrow; arrowheads depict BrdU-positive, Ki-67-negative cells. Scale bar = 50 µm.</p

Cerebellar abnormalities caused by <i>Nestin-Cre</i> mediated deletion of <i>BRaf</i>.

<p>(A, A’) Representative sagittal sections of P21 cerebellum stained with haematoxylin and eosin (HE) are shown in the upper panel. (B,B’) HE-stained pictures display the reduced size of lobe X with disorganized glomeruli. (C, C’) Calbindin staining was used to visualize the elongated primary dendrite and the reduced and irregular arborization of Purkinje neurons in the molecular layer. Scale bars; 25 µm or as indicated. (D) Quantification of cerebellar lobule length in LV. Comparable Nissl stained slices were analysed from P21 ctrl and cKO mice. Data are mean ±s.e.m.; n = 3. (E) Glomeruli/granule cell distribution in cerebellar lobe LX. Glomeruli distribution was analysed in a defined area in three different positions of comparable slices of P21 ctrl and cKO mice. Data are mean ±s.e.m.; n = 7.</p

<i>Nestin-Cre</i> mediated deletion of <i>BRaf</i> causes postnatal death and abnormal behaviour.

<p>(A) Kaplan-Meier survival curves of mice with <i>Nestin-Cre</i> driven <i>BRaf</i> deletion. Mice were monitored daily. CKO, n = 13; ctrl mice, n = 10. (B) Abnormal behaviour of P21 cKO mice, indicated by autoaggression was observed in 13 out of 15 cKO mice. (C) Quantification of fraction of animals capable to balance on a small rod. CKO, n = 13; ctrl mice, n = 11.</p

Effects of DACE on TNFα-mediated activation of signaling pathways.

<p>(A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.</p

Effects of DACE on cell cycle arrest and apoptosis.

<p>(A) A549 cells (5x10⁵) were treated with DACE and analyzed 24h later by flow cytometry. The values indicate the percentage of A549 cells in the indicated phases of the cell cycle (sub-G0/G1, G0/G1, S and G2/M). *<i>p</i><0.001 and **<i>p</i><0.0001 as compared with control. (B) The A549 cells were treated for 12h with DACE, stained with Annexin V/PI, and submitted to flow cytometry for analysis of the apoptotic cell proportion. *<i>p</i><0.05 as compared with control. (C) The A549 cells were either untreated or treated with 0.5μM and 1.0μM DACE for 12h, fixed, stained with Hoechst and TRITC-labeled-phalloidin and analyzed by confocal microscopy. Overlay images are shown. (D) The A549 cells were treated for 12h with DACE and their cytosolic fraction was analyzed for changes in the activity of caspase-3. *<i>p</i><0.05 as compared with control. (E) A549 cells were treated with DACE for 12h and then subjected to Western blotting using antibodies as indicated. Equal protein loading was confirmed by probing for beta-actin. Representative images of three independently repeated experiments are shown. The values represent means of three independent experiments and SD.</p

Effects of DACE on c-RAF-1-induced lung tumor growth in mice.

<p>(A and B) c-RAF-1-BxB transgenic mice were injected daily with either DMSO (n = 4, A) or 1 mg/kg of DACE (n = 4, B). On day 21, the lungs were fixed, embedded into paraffin and stained for H&E (left images) or for human c-RAF-1 protein (right images). Bars, 1000 μm for upper images and 100 μm for lower images. Arrow heads on upper images indicate lung tumor areas shown on lower images. (C) The total amount of tumor tissue in the lungs of untreated control mice (<i>n =</i> 4) and DACE-treated mice (<i>n =</i> 4) measured after immunohistochemistry. The lungs of treated mice exhibited 58% (*<i>p</i><0.05, <i>t</i> test) less tumor tissue in comparison to untreated control animals. (D) Western blotting of tumor lysates of untreated control mice (<i>n</i> = 4) and DACE-treated mice (<i>n</i> = 4) for c-RAF-1-BxB expression. Beta-actin was used as a loading control. (E) Densitometric quantitation of the human c-RAF-1-BxB protein expressed in the lungs of untreated and DACE-treated mice. The lungs of treated mice exhibited 66% (*<i>p</i><0.05, <i>t</i> test) less c-RAF-1-BxB expressed protein in comparison to untreated control animals. (G) Total RNA was isolated from the lungs of untreated mice (<i>n =</i> 4) and DACE-treated mice (<i>n</i> = 4), reverse transcribed, and the expression of c-RAF-1-BxB mRNA was determined by quantitative real-time PCR. The expression of c-RAF-1-BxB mRNA was reduced by 37% after systemic treatment with DACE, albeit the means are not statistically significant when compared by <i>t</i> test. (<i>p</i>>0.05). Relationship between tumor tissue amount and c-RAF-1-BxB protein (F) and c-RAF-1-BxB mRNA (H) in lungs of all mice analyzed. The relative amounts of tumors, and c-RAF-1-BxB protein and mRNA, were measured by immunohistochemistry, Western blotting, and qRT-PCR as shown in (A/B, D and G).</p

Scheme for preparation of a novel semisynthetic derivative of cucurbitacin B (DACE).

<p>Scheme for preparation of a novel semisynthetic derivative of cucurbitacin B (DACE).</p