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. 2017 May 5;45(13):7722–7735. doi: 10.1093/nar/gkx377

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — CHD1 depletion impairs osteoblast differentiation in MSC and hFOB 1.19. (A) Osteoblast-differentiated mesenchymal stem cells (Ob. diff) transfected with control (siCon) or CHD1 (siCHD1) siRNAs were stained for alkaline phosphatase (AP) activity. Mean values ± SD (n = 2), with P-value shown. qRT-PCR gene expression analysis of CHD1 and the osteoblast-specific genes ALPL, BGLAP, RUNX2 (B) and DKK1, ELN, CTGF (C) in undifferentiated (Undiff.) and osteoblast-differentiated (Ob. diff) mesenchymal stem cells transfected with control (siCon) or CHD1 targeting siRNA (siCHD1). Values were normalized to 18S rRNA and quantified relative to control undifferentiated samples. Mean ± SD, (n = 3) (*P-value < 0.05; **P-value < 0.01; ***P-value < 0.001). (D) Heatmaps depict log2-fold changes of gene expression in MSC and human fetal osteoblasts (hFOB 1.19) during osteoblast differentiation. Relative changes upon siCHD1 treatment (Diff. +siCHD1) compared to differentiation-associated changes in gene expression are shown in the lower panel of each heatmap. Genes were sorted by log₂-fold changes with siCHD1 treatment and differentiation-regulated genes were selected by adjusted P-value < 0.05 and ±0.5 log₂-fold changes. (E) Representative H&E stained tissue sections of ectopic bone formation (pink area; see black arrow) in SCID mice implanted with MSC stably expressing a non-targeting (#nt) or CHD1-targeting shRNAs (#1, #2). Mice were sacrificed 8 weeks after implantation. Scale bars represent 100 μM. (F) Boxplots show the quantification of ectopic bone formation of control and CHD1-shRNA expressing MSC with each point representing one biological replicate. The ratio of bone area (BA) over total area (TA) was compared with Tukey multiple comparison test with a threshold at 0.05 (*). (G) Representative images of Alizarin Red S staining of MSC differentiated for 14 days in control (#nt) and CHD1-depleted (CHD1 #1, #2) are shown with the quantification of the relative percentage of stained area over total area (n = 3; * P-value < 0.05).