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. 2004 Oct;15(10):4544–4555. doi: 10.1091/mbc.E03-11-0816

Figure 2.

Figure 2.

GSK-3 inhibition restores differentiation of insulin-dependent C2ind in the presence of serum and cooperates with insulin in serum-free conditions. (A) C2ind cells were cultured in a 10% serum growth medium containing 10-7 M dexamethasone and transfected with plasmids coding for different forms of PKBβ/Akt2: wild-type (PKBβWT) and constitutively active (PKBβm) (Vandromme et al., 2001). Then, the medium was changed for a differentiation medium containing 3% serum for 3 d. Cells were harvested and analyzed by Western blot for expression of differentiation markers. (B) C2ind cells were cultured in a 10% serum growth medium containing 10-7 M dexamethasone for 3 d before induction of differentiation in a 3% serum medium for 3 d. Increasing concentrations of LiCl (5, 10, or 25 mM) or NaCl (25 mM) as a control, were added to the differentiation medium and cells subsequently harvested and analyzed by Western blot or fixed for immunofluorescence analysis. In both cases, expression of myogenin was evaluated as markers of differentiation of the C2ind and shown for the Western blot analysis is the expression troponin T a late marker of differentiation. (C) C2ind cells were cultured in a 10% serum growth medium containing 10-7 M dexamethasone for 3 d, and then the medium was changed for serum-free DMEM. C2ind cells were stimulated with LiCl (Li), insulin (i), or a combination of both and in parallel with SB216763 at 3 μM (SB) and a combination of SB216763 and insulin for 24 h before harvesting and Western blot analysis for myogenin expression.