Fig. 4

Inhibition of Kdm6a and Kdm6b activity suppresses osteogenic differentiation of Twist-1^del/+ calvarial cells. a Western blot analysis of nuclear extracts isolated from Twist-1^del/+ calvaria cells treated with GSK-J4 (1μM, 2 μM) or 0.1% DMSO vehicle control for 24 h to assess H3K27me3 levels relative to histone 4 (H4). b Real-time qPCR analysis of Runx2 and Alkaline Phosphatase (Alk Phos) transcript levels in Twist-1^del/+ calvaria cells under osteogenic inductions for 24 h. 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. c Representative images of alkaline phosphatase staining and d quantitation of alkaline phosphatase activity relative to total protein for Twist-1^del/+ calvaria cells treated with either 1 μM or 2 μM GSK-J4 or 0.1% DMSO, following 1 week of osteogenic induction. Data represent mean ± S.E., *p < 0.05, one-way ANOVA with Tukey’s multiple comparisons, n = 4 Twist-1^del/+ mice. Chromatin immunoprecipitation (ChIP) analysis of H3K27me3 levels on the transcriptional start sites of e Runx2 and f Alk Phos for Twist-1^del/+ calvarial cells cultured under normal growth media (NGM) or osteogenic inductive conditions (Osteo) for 1 week in the presence of either GSK-J4 (1 μM) or 0.1% DMSO. ChIP was performed using either IgG control antibody (IgG) or H3K27me3-specific antibody (K27me). Enriched genomic DNA was used to amplify the transcription start site of target genes. Data represent mean fold enrichment relative to input DNA ± S.E., *p < 0.05, one-way ANOVA with Tukey’s multiple comparisons, n = 4 Twist-1^del/+ mice