Power masher 2

Cells and CAM tumors were lysed in a 2× Cell lysis buffer (Cell signaling technology, Danvers, MA, USA) and then an equal amount of 2× sample buffer was added. CAM tumor was homogenized by Power masherII (Nippi, Inc., Tokyo, Japan) before adding the lysis buffer. After sonication, the lysate was denatured at 100 °C for 5 min. Target proteins were separated and detected with the Jess^TM Simple Western system (Protein Simple, San Jose, CA, USA).

The frozen liver samples were thawed and a RIPA buffer containing Halt^TM Protease Inhibitor Cocktail was added to the samples at a volume of 4 mL per 1 g wet tissue weight. The tissue samples were homogenized using a Power Masher II and Bio Masher SP (Nippi, Tokyo, Japan). The homogenates were centrifuged at 13,000 g for 5 min at 4 °C and the supernatants were obtained for western blotting. The protein concentration of each sample was measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The samples of the supernatants mixed with EzApply (ATTO, Tokyo, Japan) were heated at 95 °C for 5 min and applied to 10% SDS polyacrylamide gels, and then the separated proteins were transferred to nitrocellulose membranes (Bio-Rad). After blocking for 30 min at room temperature with 3% skim milk in phosphate-buffered saline containing 0.05% Tween 20, the membranes were incubated overnight at 4 °C with rabbit antibodies against GPx-1, GPx-4 (1:1000, Abcam, Cambridge, UK) and β-actin (1:1000, Cell Signaling, Danvers, MA, USA) diluted by Can Get Signal I (TOYOBO Co. Ltd., Osaka, Japan). The membranes were subsequently incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit IgG antibody (1:5000, Cell Signaling) diluted by Can Get Signal II (TOYOBO Co. Ltd.). Immune complexes were visualized by an enhanced Immobilon™ Western (Merck Millipore, Bedford, MA, USA).

Liriomyza trifolii were provided by Applied Entomology Laboratory, Faculty of Agriculture, Shizuoka University. The flies were isolated in Hamamatsu, Shizuoka, Japan in 1991, and maintained on the leaves of kidney bean plants in a 34 cm (width) × 35 cm (length) × 34 cm (height) cage with light–dark regime (16:8) at 23°C (Tagami et al., 2006a (link); Hidayanti et al., 2022 (link)).
Total genomic DNA was extracted from L. trifolii using Qiagen DNeasy Blood and Tissue Kit, following the manufacturer’s instruction (Qiagen, Hilden, Germany), with slight modifications. Adults insects (n = 30–50) were crushed in 50 μL ATL buffer with a motorized pestle (Power Masher II; Nippi, Tokyo, Japan); 130 μL of ATL buffer, and 20 μL proteinase-K (20 mg/mL) were added to the homogenate and incubated in a 56°C dry bath incubator (Major Science, Taiwan) for 2–3 h; 200 μL of buffer AL and 200 μL of 99.5% ethanol were added following incubation at 56°C; to maximize DNA yield, the DNA was eluted in 50 μL of buffer AE. DNA yield and purity were verified with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The extracted DNA was aliquoted for sequencing and cloning of 16S rRNA gene and the Wolbachia surface protein (wsp) gene, and metagenomic library construction for next-generation sequencing (NGS).

For quantitatively measuring collagen in the left lungs of mice, the Hydroxyproline assay was performed according to the manufacturer's instructions (Chondrex, 6017). Mouse left lung tissues (10 mg) were homogenized in 100 μl of distilled water using PowerMasher II (Nippi, 891300) with BioMasherII (Nippi, 320103).

HIA was performed according to a previous paper describing the determination of indole concentration (21 (link)). Briefly, gut content samples were diluted with 70% ethanol to 100 mg/ml and disrupted by a Power Masher II (Nippi, Tokyo, Japan). After centrifugation, supernatants were filtered using a Millipore Ultrafree MC PLHCC centrifugal filter (Merck Millipore, Billerica, MA, USA). Samples were incubated for 15 min at room temperature with 5M NaOH (Wako, Osaka, Japan) and 50 μl of 0.3 M hydroxylamine hydrochloride (Wako) in a microtiter plate. Following incubation, 125 μl of 2.5 M H₂SO₄ (Wako) was added and incubated at room temperature for 30 min. The plates were immediately read at 530 nm using optical density readings by a microplate reader.