Fig 2.

(A) Cerulenin prevents the inhibitory effect of adipocytes on osteoblasts survival– Supernatants were collected from the osteoblasts side of the membrane in the six different conditions (AD/OB, OB/AD, -/OB with and without cerulenin). Human osteoblasts were cultured for 24 hrs in 96-well plates. At 24 hrs, media were replaced with the supernatants and cell proliferation was measured at timed intervals (24–96 hrs). Cell proliferation was significantly decreased in osteoblasts cultured with the supernatant from the AD/OB untreated cells after 48, 72 and 96 hrs in culture (*P < 0.001). In contrast, osteoblasts exposed to supernatants from all other conditions either treated or untreated with cerulenin showed a progressive increase in cell proliferation at all timed intervals. (B–F) Inhibition of FA synthase prevents osteoblast apoptosis induced by adipocyte-secreted factors – Human osteoblasts were cultured in 2-well slides for 24 hrs. After 24 hrs, media were replaced with media obtained from normal confluent human adipocytes treated with either cerulenin (10 nM) or vehicle alone. After 72 hrs in culture, media were removed and cells showing apoptotic cells were identified (B–E) and quantified (F) using TUNEL assay. The TUNEL assay was able to detect a higher percentage of cells with DNA fragmentation (arrows) in the NHOst treated with supernatants obtained from untreated (B, D and F) as compared with cerulenin-treated (C, E and F) adipocytes (F) (*P < 0.01). (B and C: scale bar 100 μm), (D and E: 10 μm). (G) Caspase-3 and -7 activity was assayed using the Caspase-Glo luminescence assay and data represent the mean values (S.D.) of triplicate cultures. NHOst treated with supernatants obtained from untreated adipocytes (AD/OB-) showed higher caspase 3/7 activity as compared with cerulenin-treated adipocytes (*P < 0.001).