Figure 3. FAM57B2 functional characterization.

(A and B) Ceramide synthase assay. Lysates from HEK293 cells transfected with Fam57b2 or empty vector (A) were used in enzyme assays. Products were separated by TLC as described in Methods. Arrows indicate lipid standards. Results are representative of 3 independent experiments. 25D, 25(OH)D₃; 24R25D, 24R,25(OH)₂D₃; 24S25D, 24S,25(OH)₂D₃; 1,25D, 1,25(OH)₂D₃; 1,24,25D, 1,24,25(OH)₃D₃; M, lipid markers: PE, LacCer, PC, and Sp. (C–F) Static morphometry in control (fl/fl WT) and chondrocyte-specific mutant (fl/fl Cre) Fam57b-floxed mice. Bone length (C) and trabecular bone volume (D), number (E), and thickness (F) were assessed by micro-CT. P > 0.05, by 2-tailed t test for C–F. (G) Bone stiffness was calculated from the 3PBT. P > 0.05, by 2-tailed t test. (H) Fam57b2 expression in day-18 fracture callus from control or Cyp24a1^–/– mice supplemented or not with 50 μg/kg daily C18-LacCer. *P < 0.05, by 2-way ANOVA followed by Bonferroni’s post test. (I) Fam57b2 expression in primary chondrocytes from control or Cyp24a1^–/– mice, with or without a 24-hour exposure to 1 μM LacCer. **P < 0.01 and ***P < 0.001, by 2-way ANOVA followed by Bonferroni’s post test (n = 6). (J) Callus volume quantification was determined by micro-CT. *P < 0.05 and **P < 0.01, by 2-way ANOVA followed by Bonferroni’s post test. (K) X-ray projections of callus of chondrocyte-specific Fam57b-deficient mice on day 14 and day 18 after osteotomy. fl/fl WT, control Fam57b mice; fl/fl Cre, chondrocyte-specific Fam57b-deficient mice. The number of animals per group is indicated in parentheses.