TGF‐β1 and Sema3A are downregulated in vivo by increasing VEGF doses Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones.

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TGF‐β1 and Sema3A are downregulated in vivo by increasing VEGF doses Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl). TGF‐β1 and Sema3A are downregulated in vivo by increasing VEGF doses Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl). Gene expression of Pdgfb, Ang1, Ang2, Tgfb1, and Sema3a was quantified by qRT‐PCR and expressed as fold‐change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one‐way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF‐β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179.Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses (n = 4). The right panels represent higher‐magnification views of the left panels, which encompass the majority of the implantation sites. Scale bar = 50 μm.In situ hybridization for Sema3A (green) and CD31 (red) mRNA, with nuclei staining by DAPI, on frozen muscle sections. The three lower panels represent a higher‐magnification of the area in the white square in the top panel, as a merged image and as individual channels: arrows indicate cells expressing both transcripts (yellow), only Sema3A (green), or only CD31 (red). Scale bar = 30 μm.Quantification of the percentage of endothelial cells (CD31+) that express Sema3A (left group) and of Sema3A‐expressing cells that are endothelial (CD31+, right group). Data represent the mean ± SEM of 12 individual fields of view from three independent muscles (15–94 nuclei/image, 642 total nuclei). No statistics was applied, as data represent complementary sets.Source data are available online for this figure. Elena Groppa et al. EMBO Mol Med. 2015;7:1366-1384 © as stated in the article, figure or figure legend