Validation of GFP and BMP-2 expression in lentivirus-transduced neonatal human skin fibroblasts. Validation of GFP and BMP-2 expression in lentivirus-transduced.

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Validation of GFP and BMP-2 expression in lentivirus-transduced neonatal human skin fibroblasts. Validation of GFP and BMP-2 expression in lentivirus-transduced neonatal human skin fibroblasts. (A) Immunofluorescence analysis for GFP in vector-transduced control fibroblasts (F_GFP) and BMP-2–transduced fibroblasts (F_BMP2) in culture. Scale bar = 50 μm. (B) qPCR analysis of BMP-2 expression in control GFP (F_GFP) and BMP-2 (F_BMP2)–transduced fibroblasts, confirming expression of BMP-2 transcripts in the latter. Data expressed as mean ± SD from triplicate experiments. Statistical analysis performed using unpaired t test, **P < 0.01. (C–F) Immunofluorescence staining for BMP-2 in brefeldin-treated pericytes (C, D) or untreated pericytes (E, F), stained with anti-BMP-2 polyclonal antibody (C, E) or isotype control polyclonal antibody (D, F), confirming BMP-2 expression in pericytes (C). (G–J) Immunofluorescence staining with anti-BMP-2 (G, I) or isotype control antibody (H, J) in brefeldin A–treated F_GFP fibroblasts (G, H) and F_BMP2 fibroblasts (I, J). (K) Quantitation of BMP-2 immunofluorescence signal in F_BMP2 (n = 43 cells) and F_GFP (n = 83 cells) fibroblasts versus pericytes (n = 28 cells) in a representative experiment from a total of three experiments. Statistical significance was established by one-way ANOVA Tukey's multiple comparison test **P < 0.01, ****P < 0.0001. (L) OCs generated with F_GFP control or F_BMP2 fibroblasts stained for K15, demonstrating its absence. Dotted line indicates dermo-epidermal junction. Staining representative of three independent OC experiments. Scale bar = 100 μm. Lizhe Zhuang et al. LSA 2018;1:e201700009 © 2018 Zhuang et al.