Figure 7.

LIF mediates the effects of PKIγ deletion on osteogenesis and adipogenesis in MEFs (G). MEFs from wild type (WT) and PKIγ^−/− (KO) mice were incubated for 1 day (A), 4 days (B–D), or 14 days (E–F) in mixed osteogenic/adipogenic medium. Culture medium was supplemented with 0.25 mM 3-isobutyl-1-methylxanthine (IBMX), 1 uM forskolin (FSK), or 0.1% DMSO (C) as a vehicle control (A–B). Culture medium was supplemented with IBMX plus the indicated concentrations of the neutralizing anti-murine LIF antibody (LIF Ab) in (C) and 0.3 ug/ml of either control immunoglobulin (IgG) or the LIF antibody (LIF Ab) in (D–F). LIF protein in the cell culture supernatants was measured by ELISA (A). Osteoblast differentiation was assessed by examining alkaline phosphatase (ALP) biochemically (B–D) and cytochemically (lower panel in D) and by Alizarin Red S staining of mineral (E). Adipocyte differentiation was assessed by examining Oil Red O staining (F). * denotes p < 0.05 and NS denotes not significant. In (A–B, D–F), the horizontal bars represent the means of the 3 independent experiments indicated by the individual symbols that represent means of 3 culture wells/group, each assayed in triplicate. In (C), the symbols represent the mean ± SD of 3 independent experiments, each consisting of 3 culture wells/group, each assayed in triplicate.