Fig 1: Ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) is expressed at sites of bone formation and is upregulated during mineralization in vitro. (A) Immunohistochemistry using an anti-ENPP6 primary antibody in 4-week tibias shows expression in cells at mineralizing surfaces in both trabecular and cortical bone, including osteoblasts (black arrowheads) and early osteocytes (white arrowheads). Scale bars = 50 µm. (B) Immunofluoresence staining for ENPP6 in Enpp6 +/+ 4-week tibias shows colocalization with mineralizing markers tissue nonspecific alkaline phosphatase (TNAP) and orphan phosphatase 1 (PHOSPHO1) in both trabecular and cortical bone. ENPP6 staining is evident in both nuclei (white arrowheads) and the cytoplasm (white arrows). Scale bar = 50 µm. (C) Alizarin red staining in primary osteoblasts showing mineralized nodules generated over a time course. (D) Gene expression analysis in primary osteoblasts shows upregulation of Enpp6 over a mineralizing time course along with other mineralization markers including Phospho1 and Alpl. n = 3 (day 0); n = 4 (day 7–day 21). **p > 0.01; ***p > 0.001.