Fig. 1. Dex-mediated cell death is inhibited by FK506. INS-1 cells were treated under standard culture conditions, i.e. 11 mmol/l glucose for 1d (A and B) and 4d (C) as described in Research design and methods. (A) Representative pictures are shown for TUNEL staining of INS-1 cells of n = 3 independent experiments. (B and C) Quantification of dead cells by the amount of condensed nuclei. Results are presented as mean ± SEM for n = 3 independent experiments. denotes significant effect to control, § to dex.

Fig. 2. Dex does not stimulate [Ca²⁺]_i. INS-1 cells were loaded with fura-2 AM and cytosolic [Ca²⁺]_i measured as described under Research design and methods. Concentrations used were: 100 nmol/l dex, and 1 µmol/l CCh (carbamylcholine) in the presence of 0.5 mmol/l glucose and 11 mmol/l glucose. Shown are 4 representative measurements out of 24 from 3 independent experiments.

Fig. 3. Glucose potentiates dex-induced cell death. Cells were cultured at different glucose concentrations and 100 nmol/l dex for 4d. The percentage of condensed nuclei is presented as mean ± SEM of n = 3 independent experiments. denotes significant effect to the respective control, § to dex at 2 mmol/l glucose, # to dex at 11, 5 and 2 mmol/l glucose.

Fig. 4. Dex activated calcineurin at submaximal Ca²⁺ concentrations without changes in v_max and K_m for the substrate. Calcineurin measurements were performed with cell homogenates from control and dex-treated cells as described under Research design and methods. (A) Calcineurin activity at maximal substrate and Ca²⁺ concentrations is expressed as % of total counts added to the reaction mixture. The cells were treated with dex, RU486 and FK506 for 1d as indicated. The results are expressed as mean ± SEM. The number of independent experiments is given in each column. Dependency of calcineurin activity on substrate (B) and on Ca²⁺ (C) concentrations. The results are expressed as mean ± SEM of n = 3 (for B) and n = 4 (for C) independent experiments. significantly different to the enzyme activity of control cells at the same Ca²⁺ concentration; # to enzyme activity in the absence of FK506.

Fig. 5. Cell death induced by dex does not depend on activation of calcineurin by calpain. INS-1 cells were treated as described under Research design and methods for 1d. (A) The percentage of condensed nuclei is presented as mean ± SEM of n = 3 independent experiments. The substances were added as indicated. ALLN, N-Acetyl-Leu-Leu-Nle-CHO. (B) Representative Western blot for calcineurin out of 3 independent experiments. denotes significant effect against control.

Fig. 6. Dex treatment of cells increased the amount of Hsp90 and its interaction with calcineurin. (A) Representative Western blots for calcineurin A and Hsp90 and the quantification of n = 4 independent experiments. Results are presented as mean ± SEM. denotes significant effect against control. (B) Representative Western blots for HSP90 and calcineurin B after immunoprecipitation with calcineurin B antibodies (anti-CnB Ab) and (C) quantification of 4 independent experiments using a Molecular Imager Gel Doc System (Biorad Laboratories).