P8074

Stock conidia suspensions of B. bassiana Delta native NI8 strain was obtained from the USDA-ARS, Southern Insect Management Research Unit (SIMRU), Stoneville, MS, US. Each conidia suspension serially diluted and suspended in 50 mL of 0.04% Tween-80 (Sigma-Aldrich P8074, Darmstadt, Germany) came labeled as follows: 95% spore germination and 5 × 10⁴ (380 spores/mm²), 5 × 10⁵ (38 spores/mm²), 7 × 10⁶ (3.8 spores/mm²), 7 × 10⁷ (0.38 spores/mm²). The stock conidia suspensions were kept in an incubator at −4 °C (Percival Scientific I-36LL, Perry, IA, USA).

To determine if lulworthinone forms colloidal aggregates that affect its antimicrobial activity, an MIC assay including a non-ionic detergent was used. The antibacterial activity of a colloidal aggregate should be heavily attenuated in the presence of non-ionic detergents [24 (link),51 (link)]. An MIC assay was performed according to CLSI guidelines [48 ] using S. aureus ATCC 25923. The MIC values used are from the previous study [10 (link)]. Overnight cultures were grown in MHB (275730, BD Difco, Le Pont de Claix, France) at 37 ${}^{\circ }$ C. Two-fold dilution series of lulworthinone ranging from 128 µg/mL to 0.25 µg/mL with or without 0.025% (v/v) Tween 80 (P8074, Sigma-Aldrich, Saint Louis, MO, USA) were tested.
Assay was conducted in 96-well plates (Nunclon $\Delta$ 734-2073, VWR, Radnor, PA, USA). OD₆₀₀ values were recorded by a plate reader (Victor multilabel counter, PerkinElmer, MA, USA) at 37 ${}^{\circ }$ C for 24 h. Each test run included a growth control (media and inoculum) and a sterility control (media and water), and for quality assurance, S. aureus ATCC 25923 was tested against gentamicin (A2712, VWR, Radnor, PA, USA). Tests were performed in triplicates with three technical replicates; median MIC values are displayed.

Remodelin toxicity was assessed by the Crown Biosciences on 12 6-week-old C57BL/6N female mice, with a BW of 20 g on average (see Table S2. Animal supplier: Shanghai Laboratory Animal Center SLAC, Shanghai, China). At WTSI, Remodelin and Remodelin Fluor were dissolved in a solution of 20% DMSO, 65% (45% 2-Hydroxypropyl-b-cyclodextrin solution, H5784 Sigma Aldrich) and 15% Tween 80 (P8074 Sigma Aldrich). The vehicle-treated animals were given this solution alone, without Remodelin. Remodelin and Remodelin Fluor were administered daily by oral gavage at 100 mg per kg per day and 50 mg per kg per day respectively (defined as non-toxic doses in toxicity studies), from day 21 and until culled. Animals have been killed by CO₂ inhalation followed by cervical dislocation.