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. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: Nat Cell Biol. 2016 Aug 1;18(9):917–929. doi: 10.1038/ncb3394

Figure 3. Osteogenic Defects in Osterix-Cre-Targeted Conditional Knockouts of Snail/Slug.

Figure 3

(a) Gross view of 3-month old Snailf/f/Slug+/+/Osterix-Cre, Snailf/f/Slug−/−, Snailf/f/Slug−/−/Osterix-Cre mice and a control littermate (Snailf/f/Slug+/+). Results are representative of 5 experiments performed.

(b) Alizarin Red/Alcian Blue staining of skulls isolated from neonatal Snailf/f/Slug+/+, Snailf/f/Slug+/+/Osterix-Cre, Snailf/f/Slug−/− and Snailf/f/Slug−/−/Osterix-Cre mice. Scale bar: 2 mm. Results are representative of 3 experiments performed.

(c) Femur histology of 3-month old Snailf/f/Slug+/+, Snailf/f/Slug+/+/Osterix-Cre, Snailf/f/Slug−/− and Snailf/f/Slug−/−/Osterix-Cre mice. Scale bar: 1 mm. Results are representative of 5 experiments performed.

(d) Microcomputed tomography of the proximal femur of 12-wk old Snailf/f/Slug+/+ and Snailf/f/Slug−/−/Osterix-Cre mice. Results are representative of 10 experiments performed.

(e) Bone Mineral Density (BMD), Bone Volume/Tissue Volume (BV/TV), Trabecular Thickness (Tb.Th), Trabecular Number (Tb.N) and Trabecular Separation (Tb.Sp) were measured by microcomputed tomography in 12-wk old Snailf/f/Slug+/+ and Snailf/f/Slug−/−/Osterix-Cre mice (mean ± s.d., n=10 mice). **p<0.01; unpaired t-test.

(f) Ki67 expression in proximal femurs isolated from neonatal Snailf/f/Slug+/+ and Snailf/f/Slug−/−/Osterix-Cre mice. Scale bar: 100 μm. Results are representative of 5 experiments performed.

(g) Quantification of Ki67-positive cells from (f) (mean ± s.d., n=5 mice). **p<0.01; unpaired t-test.

(h) Relative mRNA expression of osteogenic markers in neonatal calvarial RNA extracts from Snailf/f/Slug+/+ and Snailf/f/Slug−/−/Osterix-Cre mice (mean ± s.d., n=5 mice). **p<0.01; unpaired t-test.

(i) Western blot of Snail and Slug expression in SSCs isolated from Snailf/f/Slug+/− or Snailf/f/Slug+/−/Osterix-Cre mice cultured with doxycycline (Doc; 1.5 μg/ml) under control or osteogenic conditions for the indicated time periods. Results are representative of 3 experiments performed.

(j) SSCs from (i) were cultured under osteogenic conditions for 14 d and stained with Alizarin Red S. Results are representative of 3 experiments performed.

(k) Relative mRNA expression of osteogenic markers in cultures from (j) (mean ± s.d., n=3 independent experiments). **p<0.01; unpaired t-test.