FIG. 6.

siRNA knockdown of EZH2 and MX1 or FHL1 rescued aberrant osteogenic differentiation. MSC were treated with 12 pmol of negative control siRNA (Control), EZH2 siRNA #1 (EZH2), EZH2 siRNA # 1/MX1 siRNA #1 (EZH2/MX1), and EZH2 siRNA # 1/FHL1 siRNA #2 (EZH2/FHL1) culture under osteogenic differentiation conditions for 14 days. (A) Alizarin red staining of mineral deposits in EZH2/MX1 and EZH2/FHL1 compared to control and EZH2 siRNA-treated MSC. (B) Extracellular calcium quantitated and normalized to DNA per triplicate well (n = 3 donors) for EZH2/MX1 and EZH2/FHL1 compared to control and EZH2 siRNA-treated MSC. (C) Western blot analysis of EZH2, H3K27me3, MX1, or FHL1 protein levels in EZH2/MX1 or EZH2/FHL1 siRNA-treated MSC compared to control-treated MSC under osteogenic differentiation conditions. (D) RT-PCR analysis of MX1 gen expression levels in EZH2/MX1 siRNA-treated OB compared with control siRNA OB. (E) RT-PCR analysis of FHL1 gene expression levels in EZH2/FHL1 siRNA-treated OB compared with control siRNA OB. (F) EZH2 knockdown was confirmed by RT-PCR in EZH2, EZH2/MX1, EZH2/FHL1 siRNA-treated OB compared with control OB. (G, H) RT-PCR analysis of gene expression levels for osteogenic markers RUNX2 and OP in EZH2/MX1 and EZH2/FHL1 siRNA OB when compared to EZH2 siRNA OB. Mean values ± SEM, n = 3 independent MSC donors (*P < 0.05, B, F–H, 1-way ANOVA with Dunnett's multiple comparisons test; *P < 0.05, D and E unpaired Student's t-test). Color images available online at www.liebertpub.com/scd