Act 2u

The areas of the blastema and of the stump in sanguinarine-treated planarians and controls were determined as described in Balestrini et al., 2014. In brief, the animals were photographed at the same magnification with a stereomicroscope following ethanol-fixation, after 7 days of regeneration. The areas were measured using Nikon ACT-2U imaging software. A number of 30 regenerating fragments were analyzed for each experimental condition. The size of the pharynx after in situ hybridization with Dj-mhca was measured with the same procedure.

After H 2 O 2 treatment, conidia were harvested by centrifugation (15 min, 15 700g), washed twice with PBS solution, and fixed with 3.7% (v/v) formaldehyde for 25 min at room temperature. Fixed cells were harvested by centrifugation, washed twice with PBS, and incubated with different digestion enzyme solutions as follows: 0.15 units chitinase, 10 5 units ␤-glucuronidase, 9 mg driselase (Solution A); 0.15 units chitinase, 8.5 × 10 5 units ␤-glucuronidase, 9 mg driselase (Solution B); 1.5 units chitinase, 10 5 units ␤-glucuronidase, 9 mg driselase (Solution C); 1.5 units chitinase, 8.5 × 10 5 units ␤-glucuronidase, 9 mg driselase (Solution D); for 2, 3, 4, and 6 h, at 32 °C. All enzymes for the cell wall digestion of A. fumigatus conidia were obtained from Sigma-Aldrich. For the different time points, the efficiency of the cell wall digestion was evaluated by fluorescence microscopy by Calcofluorwhite (1 mg/mL, Sigma-Aldrich). Digested conidia were observed through an oil immersion lens (Plan Fluor 100/ 1.30) on an Eclipse E400 fluorescence microscope (Nikon) equipped with a 100 W mercury lamp. Images were captured with a Nikon DS-5Mc digital camera with built-in software for image acquisition (Nikon ACT-2U). Three independent experiments with 3 replicate samples were performed.