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Systems Genetic Analysis of Osteoblast-Lineage Cells

Figure 4

Maged1 and Pard6g are novel regulators of osteoblast proliferation and differentiation.

(A) The siRNAs M1 and M2 significantly reduced the levels of Maged1 in undifferentiated pcOBs relative to a scrambled control (SC) at 48 and 96 hours post-differentiation. (B) This resulted in similarly decreased MAGED1 protein at 48 hours post-differentiation (L = protein ladder). (C) In undifferentiated pcOBs Maged1 knockdown increased proliferation rate. (D) After four days of osteogenic differentiation Maged1 knockdown increased alkaline phosphatase activity (E) and the expression of Sp7 and Akp2. In contrast, at 14-days post-differentiation Maged1 knockdown significantly decreased (F) mineralized nodule formation as determined by (G) Alizarin Red staining and (H) quantification of nodule number. (I) The siRNAs P1 and P2 significantly reduced the levels of Pard6g in undifferentiated pcOBs relative to a scrambled control (SC) at 48 and 96 hours post-differentiation. (J) This resulted in similarly decreased PARD6G protein at 96 hours post-differentiation. (K) In undifferentiated pcOBs Pard6g knockdown increased proliferation rate. (L) After four days of osteogenic differentiation Pard6g knockdown decreased alkaline phosphatase activity (M) and the expression of Sp7, Runx2, Akp2, Col1a1, Bglap1 and Ibsp. (N) At 14-days after differentiation Pard6g knockdown decreased mineralized nodule formation as determined by (O) Alizarin Red staining and (P) quantification of nodule number. In all panels *P<0.05 and #P<0.10. The data represent mean±SEM (N = 4–6 independent experiment, except for Westerns (N = 2)).

Figure 4

doi: https://doi.org/10.1371/journal.pgen.1003150.g004