Fig 6.

Early‐life–increased high‐fat diet (HFD) does not prevent HFD‐induced epigenetic regulation of osteoclastogenesis. (A) Bone marrow cells were isolated from femur of all second euthanized mice from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups, and suspended for 48 h. Nonadherent bone marrow cells were recultured in the presence of 30 ng/ml RANKL for 5 days. Attached stromal cells were kept in culture until confluence as colony‐forming unit fibroblasts for isolation of RNA. Pictures showing osteoclast morphology from one represented well of cell cultures of all four groups after TRAPase staining. (B) Osteoclast numbers per well from cultures of nonadherent bone marrow cells from all four groups of no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. (C–F) Real‐time PCR for Ezh2, DNA methyltransferase 3a (DNMT3a), interferon regulatory factor 8 (IRF8), and nuclear factor of activated T‐cells 1 (NFATc1) mRNA expression in total RNA isolated from nonadherent hematopoietic bone marrow cells from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. (G–J) Real‐time PCR for alkaline phosphatase (ALP), osteocalcin (OC), collagen 1 (col 1), and osteopontin (OPN) mRNA expression in total RNA isolated from attached stromal cells as colony‐forming unit fibroblasts from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. Data were expressed as mean ± SD (n = 6 per group). p Value was analyzed by two‐way ANOVA with a HFD and early‐life PA as the main factors and their interactions. Additionally, * p < 0.05 by t test compared with no PA/control to other groups. HF, high fat.