Figure 3

Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin. OA osteoblasts were plated at 10,000 cells/cm² and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A) Incorporation of BrdU by OA Ob in response to leptin; B) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. Values are the mean ± SEM of at least four separate experiments; *P < 0.05, **P < 0.01.