Proteinase k

B. cereus was routinely grown at 25°C or 37°C in LB broth or on LB agar (BD, Franklin Lakes, NJ, USA) supplemented, when required, with erythromycin (5 μg mL⁻¹), tetracycline (10 μg mL⁻¹) and kanamycin (50 μg mL⁻¹). For biofilm assays Tryptic Soy Broth (TSB) (EMD Millipore, Billerica, MA, USA), sterilized by filtration (autoclaving was found to reduce biofilm formation by B. cereus 905), was used. TSB broth was supplemented, where indicated, with 1% glucose, proteinase K (0.1 mg mL⁻¹) (Omega Bio-Tek, Norcross, GA, USA) or DNaseI (5 U mL⁻¹) (Qiagen, Valencia, CA, USA). Other media utilized were TSB without glucose (Peptone from casein [BD] 17 g L⁻¹, Peptone from soymeal (Amresco, Solon, OH, USA) 3 g L⁻¹, NaCl (Sigma, St. Louis, MO, USA) 5 g L⁻¹ and dipotassium hydrogen phosphate (Macron, Center Valley, PA, USA) 2.5 g L⁻¹), BHI (EMD Millipore), MSgg (Branda et al. 2001 (link)), LBGM (Shemesh and Chai 2013 (link)) and M9 (De Kievit et al. 2001 (link)). Escherichia coli DH5α was grown at 37°C in LB medium and on LB agar supplemented, when required, with ampicillin (100 μg mL⁻¹), kanamycin (50 μg mL⁻¹) or tetracycline (10 μg mL⁻¹). The pH of biofilm cultures was measured by spotting 20–30 μL on pH-indicator strips (range 5.0–10.0) (EMD Millipore).

Decellularized scaffolds (n = 4) were first minced and then homogenized using a FastPrep 24 Tissue Homogenizer (MP Biochemicals, Santa Ana, CA, USA). The suspension was then digested with proteinase K (Omega Bio-tek, Atlanta, GA, USA) for roughly 1 h at 56 °C. After which 1 ml methylene blue solution (methylene blue 0.25 g/l, anhydrous sodium sulfate 50 g/l, concentrated sulfuric acid 10 ml/l) was added. The samples were then extracted using chloroform, and absorbance measurements were conducted at a wavelength of 650 nm using the microplate reader to quantify the SDS contents within the extracts.

S. epidermidis o/n cultures were diluted to an OD₆₀₀ of 0.05 in 200 µl of the TSB medium supplemented with 0.5% glucose and grown statically at 37 °C for 24 h in 96-well microplate (Greiner Bio-One). Supernatants were discarded, biofilms on the well bottom were washed twice with 100 µl of PBS and resuspended in 200 µl of PBS. The biomass of the resuspended biofilm was determined by OD₆₀₀ measurement. To determine matrix composition, proteinase K (Omega Bio-Tek) at 0.1 mg/ml and DNase I (Roche) at 10 U/ml were added at inoculation, and biofilm formation was quantified after 24 h with crystal violet. Therefore, wells were washed three times with 200 µl of PBS, dried at room temperature before staining with 0.1% crystal violet for 30 min. After three wash steps with distilled water, adhering dye was dissolved with 30% acetic acid, and the absorption was measured at 570 nm. To determine the effect of antibiotics on biofilm integrity antibiotic media exceeding the MICs at least 40-fold were added onto the 24 h pre-grown biofilms (20 µg/ml ciprofloxacin or 20 µg/ml ciprofloxacin and 12 µg/ml rifampicin in TSB 0.5% glucose). Biomass was determined by OD₆₀₀ measurement of the resuspended biofilm.

The expression of relevant Caco2 genes was evaluated by first washing the GelMA-alginate spheres with PBS 1X (Sigma-Aldrich, St. Louis, MO, USA) and then digesting the spheres with 30–50 μL proteinase K (Omega Bio-tek, Norcross, GA, USA) in a water bath at 50 °C for 2 h, with intermittent vortexing. The samples were then centrifuged at 10,000 RPM for 5 min, and the supernatant was recovered. The RNA extraction was conducted using the RNAeasy Mini Kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instructions. Total RNA was quantified using Nanodrop (ThermoFisher, Waltham, MA, USA), considering a 260/280 ratio of 2.0. For gene expression, we used an RT-qPCR SYBR Green kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instructions. RT-qPCR was conducted using a Rotor gene SYBR green filter (QIAGEN, Germantown, MD, USA). GAPDH was used as a housekeeping gene to estimate the relative gene expression of the vascular endothelial growth factor (VEGF-A) and E-cadherin (E-cad) using 2^−∆∆Ct method. One-way ANOVA, followed by the post hoc Bonferroni test (p values < 0.05), was used to estimate statistical differences. The primer sequences used in these RT-qPCR experiments are listed in Table 1.

To prepare and sequence whole‐genome DNA libraries of the 13 new Anastrepha specimens, we performed DNA extractions following the protocol in Dupuis, Sim, et al. (2018 (link)). We homogenized the tissue of the entire flies using FastPrep 24 homogenizer (MP Biomedical). Homogenized tissue was digested with proteinase K (Omega, BioTek) for 3–12 h at 55°C. We then extracted genomic DNA using the Mag‐Bind Tissue DNA KF Kits (Omega, BioTek) in an automatic extractor instrument (KingFisher Flex‐96, Fisher Thermo Scientific) following manufacturer's instructions with RNase A treatment. Whole‐genome resequencing (WGRS) DNA libraries (Table 1) were prepared using NEBNext Ultra II DNA Library Prep Kits for Illumina (New England BioLabs) following the manufacturer's instructions. Libraries were quantified using a Fragment Analyzer Automated Capillary Electrophoresis System with HS Genomic DNA Kit (Advanced Analytical Technology), and barcoded to be pooled into two final libraries containing six and seven individuals, respectively, which were each sequenced with pair‐end (PE) sequencing (2 × 150 bp) in an individual lane of the Illumina HiSeq X platform (Beijing Genomics Institute).