Figure 4. YAP/TAZ regulate epidermal SC differentiation through Notch inhibition.

(a,b) Mechanical forces regulate Notch signalling. nHEK cells were plated either on fibronectin-coated plastic dishes (stiff) and fibronectin-coated 1 kPa polyacrylamide hydrogels (soft) (a) or at sparse and dense culture conditions (b). After 24 (a) or 48 h (b), cells were harvested and analysed by qRT–PCR for the expression of the Notch target genes HES1, HES5, NRARP and NOTCH3. For each gene, data were normalized respectively to the stiff (a) or the sparse (b) condition (black bars). (c) F-actin-targeting drugs activate Notch signalling. nHEK cells treated for 24 h with control vehicle (Mock), 0.8 μM latrunculin A (LatA) or 2.5 μM cytochalasin D (CytoD) were analysed by qRT–PCR for the expression of the Notch target genes HES1, HES5 and NOTCH3. (a–c) Bars represent mean+s.d. (*P<0.05, **P<0.01, ***P<0.001 compared to the respective stiff (a), sparse (b) or Mock (c) controls; Student's t-test). (d,e) YAP/TAZ activity regulates Notch signalling. (d) nHEK cells infected with the empty vector (Mock) or with the indicated siRNA-insensitive doxycycline-inducible lentiviral YAP constructs were plated either on stiff or soft ECM substrates, as in Fig. 2f. Data were normalized to the Mock-infected cells plated on stiff. Bars represent mean+s.d. (*P<0.0001 compared to the Mock-infected cells on stiff, ^§P<0.0001 compared to the Mock-infected cells on soft; two-way analysis of variance (ANOVA)). (e) nHEK cells infected as in d were transfected with control (siCo.) or YAP/TAZ siRNAs (siYAP/TAZ), and 8 h posttransfection, cells were treated with doxycycline for 40 h. Cells were analysed by qRT–PCR for the induction of Notch target genes. Data were normalized to the Mock-infected cells transfected with siCo. Bars represent mean+s.d. (*P<0.001, **P<0.0001 compared to Mock-siCo; ^§P<0.001, ^§§P<0.0001 compared to the Mock-siYAP/TAZ; two-way ANOVA). (a–e) See Methods section for reproducibility of experiments.