Fig. 10. Treatment with lithium or Wnt CM leads to the accumulation of ß-crystallin protein without FGF2 pre-stimulation. (A) After treatment with 50 ng/ml FGF2, explants were cultured for 5 days in the presence of 10 mM NaCl (as a control) or 10 mM LiCl and observed by microscopy. Note that cell elongation did not occur in response to FGF2/LiCl. Scale bar: 50 µm. (B) Western blot analysis of explants cultured for 5 days in the presence of FGF2/NaCl and FGF2/LiCl showed that lithium induced a significant induction of Ser9 phospho-GSK-3ß. (C) Explants were pre-stimulated with or without 50 ng/ml FGF2 for 1 hour, and then cultured with 10 mM NaCl, 10 mM LiCl, control medium, or Wnt3a CM for 5 days, or in the presence of 50 µM U0126 or DMSO. Explants were then fixed and labeled with antibodies against ß-crystallin or Hoechst 33258 for nuclei staining. Immunolabeling for ß-crystallin in FGF2/LiCl-treated explants showed that ß-crystallin was accumulated without cell elongation (b). Lithium also induced the accumulation of ß-crystallin in explants cultured without FGF2 pre-stimulation (d). In the presence of U0126, FGF2/Wnt CM-treated explants revealed an accumulation of ß-crystallin without cell elongation (g), whereas the accumulation of ß-crystallin and cell elongation are induced in FGF2/Wnt CM-treated explants (f). In the presence of Wnt CM without FGF2 pre-stimulation, ß-crystallin was also accumulated (i). Scale bar: 10 µm.