Figure 7: SFRP1 knockout normalizes mineralization in Enpp1^asj osteoblasts.

A. Comparison of the relative Sfrp1 expression in differentiating calvarial osteoblasts derived from WT, Enpp1^asj, and Enpp1^T238A mice at day 7 and 21. B. Osteocalcin (OCN) transcription levels in calvarial osteoblasts derived from WT, Enpp1^asj, and Enpp1^T238A mice at day 21 demonstrate decreased osteogenesis in the Enpp1^asj cells. C. Quantitation of calcium phosphate via Alizarin Red in Sfrp1 knockdown WT and Sfrp1 knockdown Enpp1^asj calvarial cells demonstrates abrogation of the mineralization defect in the Enpp1^asj cells. D – E. Western blot analysis of SFRP1 and Nuclear ß-catenin verifies SFRP1 protein knockdown in WT and Enpp1^asj calvarial cells with corresponding changes in nuclear ß-catenin. markedly increases in SFRP1 knockdown cells, demonstrating an inverse correlation between SFRP1 and nuclear ß-catenin which, combined with the calcification phenotypes (Fig. 6A and 7C) supports the hypothesis that WNT inhibition by SFRP1 induces deficient mineralization in Enpp1^asj mice. p values are explicitly stated between 0.05 ≥ p ≥ 0.001. ***p<0.001, ****p<0.0001, Student’s unpaired T-test (to respective WT sibling pairs).