Fig. 2

Kdm6a and Kdm6b promote osteogenic differentiation in Twist-1^del/+ calvarial cells. a Real-time qPCR analysis of Kdm6a, Kdm6b, and Alkaline Phosphatase (Alk Phos) levels in Twist-1^del/+ calvarial cells treated with siRNAs targeting either Kdm6a (siKdm6a1 or siKdm6a2) or Kdm6b (siKdm6b1 or siKdm6b2), compared to the siRNA scrambled control (Scram), under osteogenic inductive conditions. Data represent mean gene expression levels normalized to β-actin ± S.E. expression, *p < 0.05, two-tailed, not-paired, non-parametric student’s t test, n = 3 Twist-1^del/+ mice. b Representative images of alkaline phosphatase staining of Twist-1^del/+ calvaria cells treated with siRNA Scram control or Kdm6A and Kdm6B-specific siRNA, following 1 week of osteogenic induction, scale bar = 100 μm at ×50 magnification. c Quantitative analysis of alkaline phosphatase activity relative to total protein for Twist-1^del/+ calvaria cells treated with Scram control and Kdm6a and Kdm6b-specific siRNA, following osteogenic induction. Data represent mean ± S.E., *p < 0.05, two-tailed, not-paired, non-parametric student’s t test, n = 4 Twist-1^del/+ mice. d Representative images of Alizarin Red mineral staining of Twist-1^del/+ calvaria cells treated with siRNA Scram control or Kdm6A- and Kdm6B-specific siRNA, following 2 weeks of osteogenic induction, scale bar = 100 μm at ×50 magnification. e Analysis of extracellular calcium levels relative to total DNA for Twist-1^del/+ calvaria cells treated with siRNA Scram control or Kdm6A- and Kdm6B-specific siRNA, following 2 weeks of osteogenic induction. Data represent mean ± S.E., *p < 0.05, two-tailed, not-paired, non-parametric student’s t test, n = 4 Twist-1^del/+ mice