Biomasher

Three hours after the last injection of NE inhibitor or PBS, the palatal gingival tissues were homogenized in Tris–HCl buffer using BioMasher (Nippi, Tokyo, Japan). These samples were centrifuged at 200 g for 5 min, and NE activity in the supernatant was determined by a method using the NE-specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Merck Millipore, Billerica, MA, USA), as previously described^{39 (link)}.

Synchronized worms were cultured on OP or Q plates (50 μM and 500 μM) for 96 h, before mRNA for genes of interest (Table 1) [25 (link)–30 (link)] was extracted via crushing using the Power Masher^® and Bio Masher^® (Nippi Inc., Tokyo, Japan), and reverse-transcribed to cDNA using the PrimeScript^™ RT Reagent Kit and the gDNA Eraser (Takara, Shiga, Japan). A real-time quantitative PCR (qPCR) was conducted using the Thermal Cycler Dice^® Real Time System Lite (Takara) with Thunderbird^® SYBR^® qPCR Mix (TOYOBO, Osaka, Japan), and appropriate primers (Table 2). Actin was selected as a reference gene and confirmed the suitability by comparing with tba-1 and pmp-3, used as alternative reference genes [31 (link)]. Each qPCR reaction was performed in triplicate. Assays were conducted at least three times independently.

After housing, male C67BL/6 mice were divided into three groups at random: vehicle (n = 5), 10 mg/kg OBG (n = 4) and 10 mg/kg T0901317 (n = 4). Samples were suspended in 0.5% aq. carboxymethylcellulose and DMSO (ratio = 2:1) and administered intraperitoneally (0.15 mL/10 g) once a day for 5 days. On the day after the final administration, the liver was removed under anaesthesia by pentobarbital, frozen in liquid nitrogen and stored at −80 °C until needed. Triglyceride analysis was performed as previously described^{21 (link)}. Briefly, ~100 mg of liver (wet weight) was homogenised in 0.5 mL of MeOH using a Biomasher (Nippi Inc.). The homogenised suspension was transferred to a new tube, and another 0.5 mL of MeOH was added and homogenisation was repeated five times. The entire 3 mL MeOH extract was mixed with 6 mL of CHCl₃ and incubated at RT for 30 min. The supernatant (2 mL) was dried under an N₂ stream at 40 °C, and the resultant residue was dissolved in 0.5 mL of 50% aq. DMSO. The concentration of triglycerides was measured using a Triglyceride E-test Wako kit (Wako Pure Industries, Ltd.) in accordance with the instructions.

Mouse brain tissue was disrupted with Qiazol Lysis Reagent (#79306; Qiagen) and Biomasher (#320102; Nippi, Tokyo, Japan), and mRNA was extracted using the RNeasy Plus Universal Midi Kit (#73442; Qiagen). Reverse transcription of the extracted mRNA was performed using the ReverTra Ace qPCR RT Master Mix (FSQ-201; Toyobo, Osaka, Japan). The primer sequences are shown below. Dscam Exon1 (Forward: 5’-CCCTCCTGTGACTCTCAGATG-3‘, Reverse: 5’-CCCGTAAAACAGCCTTGATGTG-3’), Dscam Exon2-3 (Forward: 5’-ATCCTGGACGGGTTTGACCA-3‘, Reverse: 5’-GCGGCTGTTCTCAATGAGC-3’), Dscam Exon15 (Forward: 5’-GTACCCAGCTACCCTCCTGA-3‘, Reverse: 5’-CTCCTTGGACAGTGTGGACC-3’), Dscam Exon21 (Forward: 5’-TCCTCCATCACCCTCTCCTG-3‘, Reverse: 5’-TTTCCCCAGGGTTTTGGCTT-3’), and β-actin (Forward: 5’-GGCTGTATTCCCCTCCATCG-3’, Reverse: 5’- CTCCTTGGACAGTGTGGACC-3’). PowerUp SYBR Green Master Mix (A25742; Applied Biosystems) was used for PCR, and amplification and detection were performed using LightCycler96 (Roche). The relative amounts of mRNA were calculated using the ΔΔCt-method. To briefly describe the calculation method, the Ct value of Dscam in each sample was ΔCt value-corrected by the Ct value of β-actin. ΔCt values were compared relative to each other (ΔΔCt value).

A colony formation assay was conducted to determine the amount of gut bacteria present in adult Drosophila. Five bacterial culture media were used: brain heart infusion (BHI) broth [18.5 g Bacto BHI (Becton Dickinson 237500), 7.5 g agar and 500 ml distilled water]; lysogeny broth [LB; 10 LB tablets with agar (Lennox, Sigma-Aldrich L7025) and 483 ml distilled water]; de Man, Rogosa and Sharpe (MRS) broth [26 g MRS broth (Oxoid CM0359), 7.5 g agar and 500 ml distilled water]; liver infusion broth [LIB; Difco LIB (Becton Dickinson 226920), 7.5 g agar and 500 ml distilled water]; and mannitol [12.5 g d-Mannitol (Sigma-Aldrich M4125), 1.5 g Bacto Peptone (Beckton Dickinson 211677), 2.5 g select yeast extract (Sigma-Aldrich Y1000), 7.5 g agar and 500 ml distilled water]. The guts of 10 HGD-treated unmated females at 5 days post-eclosion were dissected in 50 mmol l⁻¹ Tris-HCl. The dissected guts were placed in 250 µl of each liquid medium, and the tissues were mashed using a BioMasher (Nippi). The gut sample solutions were diluted 1–1/16. After 5 days of incubation, the number of colonies growing on each plate was counted, and the colony-forming units (CFU) were calculated. The number of replicates used is given in the individual figure legends.

KillerFirefly-expressing HEK293T was harvested and homogenised using BioMasher (Nippi) in a Tris-buffer (100 mM Tris-Hcl pH 8.0). Lifeact-KillerFirefly protein was transferred to a 96-well plate (Nunclon Delta Surface, Thermo Fisher) and luciferin (1 mM final) was added to each well. Spectrum data was collated using SpectraMax i3 (Molecular Devices).

For the NBT method, KillerFirefly-expressing HEK293T was treated with 2 mM luciferin and 1 mg/ml of NBT for 1 h in a CO₂ incubator. The precipitate was dissolved in DMSO and absorbance at 560 nm was measured^{30 (link)}. For the NIR-CLA method, KillerFirefly- expressing HEK293T was harvested and homogenised using BioMasher (Nippi) in PBS and treated with 2 mM luciferin and 10 µM NIR-CLA (Atto). Luminescence was measured using an Aequoria-2D/C8600 system (Hamamatsu photonics).