Fig. 5.

Effects of secreted factors and osmolytes on encystation and GSK3 activity. a Secreted factors. Wild-type and gsk3⁻ cells were grown to stationary phase with autoclaved K.aerogenes, and medium and cells were separated by centrifugation. The cells were subsequently incubated for 60 h in either water, wild-type medium or gsk3⁻ medium, and cyst percentages were determined after staining with Calcofluor. Means and SE of 4 experiments are presented. b Sorbitol. Wild-type, gsk3⁻, gsk3⁻/GSK3 and wild-type/GSK3 cells were incubated with the indicated concentrations of the osmolyte sorbitol. After 48 h of incubation, cells were stained with Calcofluor and percentages of fluorescent cysts were determined. Means and SE of 3 experiments. c Osmolyte sensitivity. Wild-type and gsk3⁻ cells were incubated in 10 mM NH₄Cl, 10 mM KCl or 20 mM glucose for 48 h. After Calcofluor staining, the percentage of fluorescent cysts was determined. Means and SE of 4 experiments. d GSK3 activity. Wild-type cells were incubated with and without 10 mM NH₄Cl and gsk3⁻ cells without NH₄Cl only. Cell extracts were prepared at the indicated time points and incubated with [γ-³²P]ATP and the GSK3 substrate phosphoglycogen synthase peptide-2 in the presence and absence of the GSK3 inhibitor LiCl. After 8 min, ³²P incorporation in the peptide was measured and non-specific ³²P incorporation in the presence of LiCl was subtracted [17]. Means and SE of 4 experiments are presented