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. 2018 Oct 15;9:1426. doi: 10.3389/fphys.2018.01426

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Copyright © 2018 Quarto, Shailendra, Meyer, Menon, Renda and Longaker.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Twist1 silencing phenocopies the osteoskeletal profile of Twist1 haploinsufficiency in POb by downregulating Fgf23. (A) Osteogenic differentiation assay performed on shTwist1 POb revealed a robust extracellular matrix mineralization. (B) Quantification of Alizarin red staining for the osteogenic assay shown in panel A. (C) RT-qPCR analysis showing significant upregulation of Bglap as compared to scramble POb. (D) RT-qPCR analysis performed on stable transduced cells upon puromicin selection reveals that sh-mediated Twist1 silencing triggers a significant downregulation of Fgf23 expression. (E) Immunoblotting analysis using anti-FGF23 antibody confirms decreased FGF23 at protein level in shTwist1 POb compared to scramble POb. (F) Bulk RNA-seq analysis showing the effect of Twist1 haploinsufficiency on some of its target genes such Zeb, Snail1, and Cdh1 and comparison in wild-type versus Twist1 haploinsufficiency parietal bones. (G) As assessed by RT-qPCR analysis a similar pattern is observed in stable transduced shTwist1 POb and scramble POb upon puromicin selection and culturing them. Values: ^∗p ≤ 0.05, ^∗∗p ≤ 0.01.