FIG. 5.

Chitin synthase activity increases in response to caspofungin in a calcineurin-dependent manner. (A) The reproducibility of the nonradioactive chitin synthase assay was verified by analysis of total chitin synthase activity in increasing amounts of starting protein (5 and 10 μg). Results were calculated as stated in Materials and Methods and are presented as the mean ± SD for total chitin (μg/ml) produced during the 90-min incubation. Total chitin synthesis in complete reaction mixture (black bars), total chitin remaining after a 5-h treatment with chitinase (1 U/ml) at room temperature (light gray bars), chitin synthesis in reaction mixture lacking UDP-GlcNAc substrate (dark gray bars), and chitin synthesis in reaction mixture lacking trypsin for activation (white bars) are shown. Experiments were performed in biological triplicate for statistical analysis. (B) Comparison of the total chitin synthase activity of the wild-type (WT) and ΔcnaA strains after exposure to either 4 μg/ml caspofungin alone (gray bars) to that of the WT strain after cotreatment with 4 μg/ml caspofungin and 200 nM FK506 (black bar). Results and error bars represent the means ± SD for chitin (μg/ml). (C) Comparison of total chitin synthase activity in WT microsomal membrane extracts pretreated with TFP (100 or 200 μM) or FK506 (200 or 400 μg/ml) for 30 min at room temperature. Results and error bars represent the means ± SD for chitin (μg/ml). All experiments were performed in biological triplicate for statistical analysis.