Journal: Scientific Reports

Article Title: Fine-tuned SRF activity controls asymmetrical neuronal outgrowth: implications for cortical migration, neural tissue lamination and circuit assembly

doi: 10.1038/srep17470

Figure Lengend Snippet: ( A ) Left panel: distribution of SRE site prediction scores (>0.8) for SRF-upregulated genes and an equal number of SRF downregulated genes. Right panel: percentage of genes with an SRE matching score >0.8 within up- and downregulated genes. ( B ) Salient GO categories enriched in SRF-upregulated genes. Depicted are the enrichment over baseline and the transformed adjusted p-value. The vertical line indicates the 0.05 significance threshold. ( C ) Scheme of the experiments. Cultures infected at DIV4 and samples collected at DIV10. ( D ) ChIP assays confirm the direct binding of caSRF to the promoter of Arc and Egr1. ChIP-qPCR assays for specific promoters using primers close to the SRE-containing region. The number of SRE sites and the highest Transfac matrix similarity score for SRF binding (obtained with PSCAN) are indicated. α-VP16: specific antibody, IgG: preimmune. *p < 0.05; **p < 0.005: ns: not significant; Student’s t test (n = 4–6). ( E ) RT-qPCR assays confirmed the upregulation (left) and downregulation (right) of candidate genes retrieved in the transcriptomics analysis. *p < 0.05, **p < 0.005 referred to GFP. Student’s t test (n = 3–4). ( F ) The co-infection of neuronal cultures with ciSRF and caSRF shows that ciSRF interferes with caSRF-dependent transcription. Data in bar graphs are expressed as means of the fold changes ± SEM and were compared using 1-way ANOVA, followed by Bonferroni’s multiple comparison test. The (*) symbols indicate statistical difference between all the conditions and caSRF; *p < 0.05; **p < 0.005; ***p < 0.0005 (n = 3–4). ( G ) Immunohistochemistry against Egr1 in cortices of P20 mice electroporated with GFP alone (GFP) or in combination with caSRF (caSRF). Scale bar: 100 μm. ( H ) GFP- and caSRF-electroporated retina showing cells with upregulated Egr1 and Arc upon caSRF expression (red arrowheads).

Article Snippet: The primary antibodies used in this study are α-GFP (Aves Labs, GFP-1020), α-MAP2 (Sigma, M9942), α-GFAP (Sigma-Aldrich, G9269), α-VP16 (mouse monoclonal from Santa Cruz Biotechnology sc-7545; rabbit polyclonal from Sigma-Aldrich V4388), α-Egr1 (Santa Cruz Biotechnology, sc-110), α-SRF (Santa Cruz Biotechnology sc-335), α-dsRed (Clontech, 632496) and α-Arc (Synaptic Systems, 156-003).

Techniques: Transformation Assay, Infection, Binding Assay, ChIP-qPCR, Quantitative RT-PCR, Comparison, Immunohistochemistry, Expressing