Pm 996

For observing AF-derived fluorescence, we prepared PDA supplemented with 3 g·L⁻¹ α-CD (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 0.3 g·L⁻¹ activated carbon (Table 1). We also prepared PD broth (Merck) with activated carbon and without α-CD as the liquid culture medium. Aliquots (495 μL) of the liquid medium were dispensed into 1-mL pipette tips that were sealed with laboratory film (PM-996, Bemis, Neenah, WI, USA) and used for incubation [27 (link)]. The total volume was adjusted to 500 μL by adding the spore solution. To prepare trace element solutions, (CH₃CHOCOO)₂Ca·5H₂O, MgSO₄·7H₂O, FeCl₃·6H₂O, and [CH₃CH(OH)COO]₃Al (FUJIFILM Wako Pure Chemical) were dissolved in distilled water. The trace element solutions were added to the liquid culture media. The final concentrations of Ca, Mg, and Fe were set to 5 mg·L⁻¹ and that of Al was set to 3 mg·L⁻¹. The fungal spore solution (5 μL) was dispensed onto a culture plate or into the liquid culture medium. After liquid cultivation, the laboratory film was removed from each pipette tip, and the tips were centrifuged at 3000× g for 15 min. The AF concentrations were measured in the obtained culture solutions. Mycelia with spores in the incubation tip were dried in a dehydrator (SLI-450N, TOKYO RIKAKIKAI, Tokyo, Japan) at 65 °C overnight, and the fungal mass was calculated using the tip masses before and after incubation.

Resected xenograft PA samples were cut to proper size by a scalpel and immobilized on 6 cm-cell culture dishes (Thermofisher, cat. no. 15462) with two slices of 1mm× 5 cm-parafilm (Bemis, PM-996). Then the tissues were immersed in 1× PBS, and applied to the AFM force-measuring setting. A homemade AFM from Institute of Biophysics, Chinese Academy of Sciences was used in this study. Using constant force mode, more than 2000 force-to-distance traces (force curves) were recorded and more than 300 traces were selected for each group. The slope K (pN/nm) of every trace was calculated and then transformed to Young’s modulus (kilopascal, kPa) by using JPK Data Processing software. The final Young’s modulus of each tumor was calculated taking into account all traces recorded for a single tissue sample. The tumor stiffness between sunitinib treatment and control group were compared by mean Young’s modulus of each group, which was calculated by the Gaussian fitting via the amplitude version of Gaussian peak function (GaussAmp) in Origin software.

To observe conidial formation, PDA plates without any additional reagents were first prepared. To observe AF-derived fluorescence, PDA supplemented with 3 g·L⁻¹ α-CD (Wako Pure Chemical Industries, Osaka, Japan) and 0.3 g·L⁻¹ activated charcoal powder (Neutral, Wako Pure Chemical Industries) was prepared. PD broth (Merck, Darmstadt, Germany) with activated carbon and without α-CD was also prepared as a liquid culture solution. Then, 495 μL of liquid medium was dispensed into 1-mL pipette tips, which were used as incubation apparatus (Figure S2), and the total amount was adjusted to 500 μL by adding the spore solution. Each spore solution (5 μL) was dispensed onto a culture plate or into the liquid culture medium. After liquid cultivation, the laboratory film (PM-996, Bemis, Neenah, WI, USA) wrapped around the top of each 1-mL tip was removed, and the tips were centrifuged at 2000× g for 10 min. The obtained culture solutions were used to measure the AF concentrations. The mycelia with spores in the incubation tips were dried in a dehydrator (SLI-450N, TOKYO RIKAKIKAI, Tokyo, Japan) at 65 °C overnight, and the fungal dry weight was measured by comparing the tip weight before and after incubation.