(C) The area, number, and mean size of adipocytes were determined by the ImageJ software. from wild-type or OPN-/- MSCs was assayed by real-time PCR. (F) Expression of OPN by Zs-OPN MSCs. Total RNA was extracted from both wild-type and Zs-OPN MSCs and OPN expression was determined by real-time PCR (left panel). Total protein was collected and OPN expression was determined by western blotting analysis (right panel). Antibody for detection of the two isoforms of OPN was purchased from Santa Cruz. sOPN, secreted OPN; iOPN, intracellular OPN. All values are means SEM. Pravastatin sodium Fig. S2. Knockdown of OPN in wild-type MSCs skews the Pravastatin sodium balance of MSC differentiation. (related to Physique 1) (A) Wild-type MSCs transfected with lentivirus expressing OPN-specific (shOPN) or control (shControl) shRNA were analyzed for OPN expression by real-time PCR (left panel) or western blotting analysis (right panel). (B and C) Cells transfected as in (A) were cultured in adipogenic differentiation medium for 6 days and stained with Oil Red O (bar: 100 m; adipocytes counted in six random fields from four cultures per group) (B), or in osteogenic differentiation medium or control medium for 17 days and stained with Alizarin Red S (bar: 500 m) (C). (D) Wild-type MSCs were cultured under the osteogenic differentiation medium with or without recombinant OPN (rmOPN, 1 g /ml) for 15 days and stained with Alizarin Red S (bar: 500 m). Values are means SEM. ***, 0.001. Representative of at least three impartial experiments. Fig. S3. Antibody neutralization of OPN significantly promoted adipogenic differentiation of MSCs. (related to Physique 3) MSCs within 15 passages were cultured in adipogenic differentiation Pravastatin sodium medium supplemented with OPN-specific monoclonal antibodies (2C5, 1H3F7, and 2A1; 1 g/ml or 10 g/ml) or isotype control (10 g/ml) and renewed after 3 days. On day 6, cells were stained with Oil Red Rabbit Polyclonal to PSMC6 O (bar: 500 m). Arrows indicated adipocytes. Representative of three impartial experiments. Fig. S4. quantification of bone mineral density and adipocytes. (related to Physique 6) (A) Tibia and femur (n=9) were harvested and connective tissue removed before fixing in 4% formaldehyde for 24 hours at room heat. Fixed bone samples were scanned by micro-CT and analyzed by CTAn software. (B) Freshly isolated tibia and femur (the ends away from the knee were opened) were fixed in 4% formaldehyde for 24 hours at room heat. The bone samples were embedded and sectioned. The 5 m undecalcified sections were stained with Goldner’s trichrome staining. Adipocytes were large oval-shaped or circular cells with a cell membrane without cytoplasmic staining, indicated by an arrow in the enlargement of the lower right quadrant (bar: 500 m). (C) The area, number, and mean size of adipocytes were determined by the ImageJ software. Values are means SEM, n=5. quantification of adipocytes and osteoblasts. (related to Physique 6) Total RNA were extracted from tibia and femur according the protocol by Carter 0.05; ***, 0.001. NIHMS531694-supplement-Supp_Fig_S1-S5.pdf (5.6M) GUID:?B8CA5832-0417-42F1-AB62-D42C972C93EB Supp Table S1. NIHMS531694-supplement-Supp_Table_S1.doc (68K) GUID:?906A3EE0-7C0F-4E4C-ADF2-025ED3CEBE18 Abstract An imbalance between normal adipogenesis and osteogenesis by mesenchymal stem cells (MSCs) has been shown to be related to various human metabolic diseases, such as obesity and osteoporosis; however, the underlying mechanisms remain Pravastatin sodium elusive. We found that the conversation between osteopontin (OPN), an arginine-glycine-aspartate-containing glycoprotein, and integrin v/1 plays a critical role in the lineage determination of MSCs. Although OPN is usually a well established marker during osteogenesis, its role in MSC differentiation is still unknown. Our study reveals that blockade of OPN function promoted strong adipogenic differentiation, while inhibiting osteogenic differentiation. Re-expression of OPN restored a normal.