Fungal growth was monitored microscopically with an Olympus CK40 microscope equipped with a Zeiss MRc digital camera and the growth rates were determined spectrophotometrically as described previously [19]. Alternatively, 2 × 103 conidia were spotted in 5 μl aliquots on appropriately supplemented agar plates. The plates were then incubated at 37°C for up to 72 h. Every 24 h, the plates were photographed and the colony

diameters were determined. All assays were performed as technical triplicates and biological duplicates. Analysis of the induction of the agsA expression by a GFP-based reporter system The A. niger reporter strain selleck chemicals llc RD6.47 carries the agsA promoter fused to a nucleus-targeted GFP (H2B::eGFP) [27]. Activation of the CWIP can be monitored by the increase in nuclear fluorescence. Analysis of the activation of the agsA promoter by 10-100 μg/ml AFPNN5353 Selleck ATM/ATR inhibitor was performed

as described in [10]. As a positive control, caspofungin at a concentration of 10 μg/ml was used. Fluorescence images were taken from coverslips observed with an Axioplan 2 microscope (Zeiss) equipped with a Sony DKC-5000 digital camera. Fluorescence staining Indirect immunofluorescence staining A. nidulans was grown over night on glass cover slips at 30°C in CM. They were further incubated for 90 min in the presence or absence (controls) of 0.2 μg/ml AFPNN5353. The samples were stained as described previously [14]

and incubated with rabbit-anti-AFPNN5353 antibody (1:2.500, Novozymes, Denmark) for at least 60 min. Immunocomplexes were detected with FITC-conjugated swine-anti-rabbit IgG (1:40, DAKO, Germany). All samples were embedded in Vectashield mounting medium (Vector Laboratories, Burlingame, USA). Microscopy was done with a Zeiss Axioplan fluorescence microscope or a Zeiss confocal laser scanning microscope as described in [14]. For incubation with latrunculin B (Sigma, Austria), samples were treated with 0.2 μg/ml AFPNN5353 and 10 μg/ml latrunculin Erythromycin B for 80 min. As a control, samples were treated with DMSO to exclude artifacts evoked by the dissolvent of latrunculin B. For detection of AFPNN5353 in the presence of elevated concentrations of CaCl2, fungi were grown in CM* medium and then treated with 0.2 μg/ml AFPNN5353 in the presence of 10 mM CaCl2 for 90 min. Analysis of membrane permeabilization and cell viability To determine if AFPNN5353 permeabilized the plasma membrane of A. niger germlings, we used a combination of propidium iodide (PI) and fluorescein diacetate (cell tracker, CMFDA green, Invitrogen) according to [48]. Twelve h old A. niger germlings were grown in Vogels medium and pretreated with the two dyes (final conc.