Melatonin m5250

Melatonin (M5250) was purchased from Sigma-Aldrich (St. Louis, MO, USA). FSH was purchased from Ningbo Second Hormone Factory (Ningbo, China). The relevant information of antibodies against VEGFA, VEGFR2, FSHR, CD34, HIF-1α, Keap1, Nrf2, HO-1, TUBA1A were listed in Table 1. Other chemical agents used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless instructed otherwise.

Melatonin (M5250), insulin, oleic acid (OA), and palmitic acid (PA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bodipy was purchased from Thermo Fisher Scientific (Waltham, MA USA). The HFD (containing 60% kcal from fat) was purchased from Beijing HFK Bioscience Co. Ltd., (Beijing, China), and the detailed composition of diet is shown in the Supplementary Table S1.

Recombinant human VEGF (100-20) was bought from PeproTech (Rocky Hill, NJ, USA). VEGFR2 (07-158), phospho-VEGFR2 (orb106137), CD133 (orb13002), and β-actin (a5441) antibodies, and melatonin (M5250) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospho-FAK (3283S) antibodies was purchased from Cell Signaling (Danvers, MA, USA). FAK (sc-1688), phospho-c-Src (sc-12928-R), c-Src (2105S), phospho-c-Jun (sc-822), c-Jun (sc-74543), phospho-p65 (sc-101752), p65 (sc-8008), CD31 (sc-18916), and CD34 (sc-74499) antibodies, as well as the angiotensin II (FAK activator; sc-363643) and c-Src activator (sc-3052), were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The NF-κB activator (prostratin; ab120880) was purchased from Abcam (Cambridge, MA, USA). PDGF-BB antibody (MBS9404630) was purchased from MyBioSource (San Diego, CA, USA). The VEGFR2 short hairpin RNA (shRNA) plasmid was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan).

Tg(UAS:FingR(PSD95)-GFP-CCR5TC-KRAB(A)-P2A-mKate2f) larvae that had been electroporated with FoxP2.A:Gal4FF (see the ‘Single-cell FingR(PSD95) expression using electroporation’ section) were kept under a 14 h–10 h light–dark cycle until 7 d.p.f., then imaged at ZT4–ZT5 (see the ‘Repeated imaging of FingR-labelled synapses’ section). Larvae were transferred to individual wells of a six-well plate containing 10 ml of sleep-promoting drugs, alone or in combination, as follows: 30 µM melatonin (M5250, Sigma-Aldrich) in 0.02% DMSO; 30 µM of clonidine hydrochloride (C7897, Sigma-Aldrich) in 0.02% DMSO; 45 µM 2-chloroadenosine (C5134, Sigma-Aldrich) in 0.02% DMSO; and 0.02% DMSO in fish water as controls^{45 (link),52 (link),60 (link),61 (link)}. Combinations of drugs were applied at the same concentrations as the single-dose conditions, maintaining the final DMSO concentration of 0.02%. Sleep induction was monitored with video-tracking (see the ‘Locomotor activity assay’ section) for 5 h, after which the drugs were removed by 2–3 careful replacements of the fish water using a transfer pipette followed by transferring the larvae individually to a new six-well plate with fresh water. The larvae were then reimaged using the Airyscan system (see the ‘Repeated imaging of FingR-labelled synapses’ section).