Osteoblast differentiation is accompanied by increases in the expression of peroxisome-related genes and proteins.
(A-D) Semiquantitative RT-PCR of genes involved in osteoblast differentiation (A), peroxisome biogenesis (B) as well as of peroxisomal enzymes and transporters (C) and Ppars (D). The mRNA level of the housekeeping gene Gapdh is included in (A). G-H. Increases in the protein level of peroxisomal membrane and matrix proteins during osteoblast differentiation were confirmed by Western blot analyses of organelle fractions from primary osteoblasts. (F) Osteoblasts after 15 days in culture were collected and subjected to differential centrifugation to obtain enriched organelle fractions (S2, P2, S3, and P3: all the details can be found in Methods, chapter 11). Fractionation quality is demonstrated by Western blots for the peroxisomal marker protein PEX14, mitochondrial marker protein UQCRC2, and the cytosolic, extracellular and vesicular marker protein OPN. (G-H) Time-dependent changes in the protein levels of mitochondrial proteins SOD2, UQCRC2 (G), the bone maturation marker OPN (G) and of peroxisomal membrane (PEX13, PEX14, ABCD3) and matrix (Thiolase, Catalase) proteins (H) are shown.