Luzindole

Amyloid β peptide 1-42 (Aβ42) from Innovagen (Lund, Sweden) was prepared according to the protocol previously used in our lab [44 (link)]. Briefly, Aβ was dissolved in dimethylsulfoxide (DMSO; Sigma-Merck, Darmstadt, Germany) as a 5 mM stock, subsequently diluted to 100 μM in a culture medium, and enriched in oligomers by aggregation at RT for 24 h, followed by at least two freeze–thaw cycles prior to use. Melatonin, EX527 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and BML-275 (Enzo Life Sciences Inc., Farmingdale, NY, USA) were dissolved in DMSO as 10 mM stocks and further diluted in a culture medium for experiments. Both EX527 and BML-275 were always added 15 min before other drugs. Luzindole (Tocris, Bristol, UK) was dissolved in DMSO as 50 mM stock and further diluted in culture medium for experiments, where it was always added 30 min before other drugs. Golgi inhibitor brefeldin-A (Thermofisher Scientific, Waltham, MA, USA) was dissolved in DMSO as a 10 mg/mL stock and added during the last 3 h of treatment.

Commercial melatonin was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Ramelteon was purchased from Selleck Chemicals (Houston, TX, USA) and luzindole was purchased from Tocris Bioscience (Bristol, UK). Test samples were prepared by dissolving melatonin and ramelteon in dimethyl sulfoxide (DMSO), and the indicated concentration was reached through dilution with Tryptic Soy Broth (TSB; Bacto^™, BD, Sparks, MD, USA) or Dulbecco’s Modified Eagles Medium (DMEM, Hyclone, Logan, UT, USA). DMSO was used as the vehicle control in the experiments. The test bacterium Porphyromonas gingivalis ATCC33277 was provided by the Shanghai Research Institute of Stomatology and Shanghai Key Laboratory of Stomatology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China) and grown in fresh TSB at 37°C under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂).

Ramelteon, (S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl] propionamide (TAK-375), was synthesized by Takeda Pharmaceutical Company (Osaka, Japan) [28] (link). H89, 2′,5′-dideoxyadenosine, and GSK4112 were purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, luzindole was obtained from Tocris Cookson (Bristol, UK). Forskolin was procured from Merck Millipore (Darmstadt, Germany).

Intraperitoneal (i.p.) melatonin injections (Sigma Aldrich) were administered daily (10 mg/kg body weight) at ZT12 for 7 consecutive days. melatonin was dissolved in 0.5% absolute ethanol and saline. Luzindole (1 mg/kg i.p., a competitive MT₁/MT₂ melatonin receptor antagonist; Tocris) was administered at ZT12 for 7 consecutive days before laparotomy under sevoflurane (Abbvie) anesthesia.

Behavioral experiments were typically performed between 51 and 55 hpf. The following drugs were used: L-cis-diltiazem (Enzo Life Sciences) in natural sea water (NSW), luzindole (Tocris), stock in DMSO, mecamylamine (Sigma), stock in 95% EtOH or NSW, and melatonin (Sigma) and acetylcholine (Sigma) solutions freshly prepared in NSW. Drugs were diluted to their final concentration in NSW; corresponding concentrations of vehicle were used as controls. Ciliary beating and arrests were imaged as described (Conzelmann et al., 2011 (link)), with a DMK 21BF04 camera (The Imaging Source) and a frame rate of 60 or 15 frames/s, respectively. A 750-nm-long pass filter was always interposed between the light source and larvae. Ciliary closure time was measured from 1-min-long movies. Behavioral data were analyzed in R. All the experiments were repeated at least twice with larvae from different embryonic batches.

Ethanol, 5-ethyl-5-(1′-methyl-propyl)-2-thiobarbiturate (Inactin^®), mecamylamine, melatonin, and SDS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Luzindole was purchased from Tocris Bioscience (Bristol, UK). Sodium phosphate dibasic dihydrate (Na₂HPO₄∙2H₂O), potassium dihydrogen phosphate (KH₂PO₄), sodium hydroxide (NaOH), and sodium chloride were purchased from Merck KGaA (Darmstadt, Germany). ⁵¹Cr-EDTA was purchased from PerkinElmer Life Sciences (Boston, MA, USA). Parecoxib (Dynastat^®) was obtained from Apoteket AB (Uppsala, Sweden).