Figure 4. Snail/Slug Regulate YAP/TAZ Levels in SSCs.

(a) YAP and TAZ expression levels in SSCs isolated from Snail^f/f/Slug^+/+ and Snail^f/f/Slug^−/− mice and transduced with adeno-GFP or Cre. Results are representative of 3 experiments performed.

(b) YAP and TAZ localization in Snail^f/f/Slug^+/+ and Snail^f/f/Slug^−/− SSCs following transduction with adeno-LacZ or Cre. Nuclei were counterstained with DAPI (blue). Scale bar: 10 μm. Results are representative of 3 experiments performed.

(c) YAP and TAZ expression levels in Snail^f/f/Slug^+/+/GFP and Snail^f/f/Slug^−/−/Cre SSCs cultured under osteogenic conditions as indicated. Results are representative of 3 experiments performed.

(d) BMP2 induces Snail/Slug and YAP/TAZ levels in osteoblast progenitors. Calvarial osteoblast progenitors were isolated from E18 mice embryos, treated with 100 ng/ml BMP2 and protein expression monitored by Western blot. Results are representative of 3 experiments performed.

(e) Deletion of Snail/Slug in osteoblast progenitors blunts the BMP2-induced up-regulation of YAP/TAZ. Calvarial osteoblast progenitors were isolated from Snail^f/f/Slug^+/+ and Snail^f/f/Slug^−/− mice and transduced with adeno-GFP or Cre. Cells were then treated with 100 ng/ml BMP2 for 2 h, and protein expression monitored by Western blot. Results are representative of 3 experiments performed.

(f) YAP and TAZ expression levels in E17.5 Snail^f/f/Slug^+/+ and Snail^f/f/Slug^−/−/Dermo1-Cre calvarial lysates. Results are representative of 5 experiments performed.

(g) YAP and TAZ expression levels in E17.5 Snail^f/f/Slug^+/+ and Snail^f/f/Slug^−/−/Osterix-Cre femur lysates. Results are representative of 5 experiments performed.

(h) CFU-F colony count from bone marrow cells isolated from YAP^f/+/TAZ^f/+ mice following transduction with lentivirus GFP or Cre expression vectors (mean ± s.d., n=5 independent experiments). **p<0.01; unpaired t-test.

(i) Ki67 expression in SSCs isolated from YAP^f/+/TAZ^f/+ mice following transduction with adeno-GFP or Cre. Scale bar: 50 μm. Results are representative of 3 experiments performed.

(j) Quantification of Ki67-positive cells from (i) (mean ± s.d., n=3 independent experiments). **p<0.01; unpaired t-test.

(k) Alizarin Red S staining of SSCs isolated from YAP^f/+/TAZ^f/+ mice following transduction with adeno-GFP or Cre and cultured under osteogenic conditions for 14 d. Results are representative of 3 experiments performed.

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