Fig 2: Establishment and differentiation into neurons of iPSC line BIONi010-C-24 with doxycycline inducible A53T a-synuclein.(A) Illustration of the doxycycline (DOX) inducible SNCA expression system: One allele contained the m2 version of the reverse TET transactivator (M2rtTA) under regulation of the CAG promoter. When doxycycline binds the tetracycline reverse transactivator (rtTA), its 3D structure changes facilitating binding to the tetracycline responsive element (TRE) leading to the expression of SNCA-A53T-HA. Neomycin (Neo) and puromycine (Puro) resistance genes enable double selection. SA = splice acceptor, 2A = self-splicing protein. (B) Western Blot analysis for total a-synuclein in pluripotent iPSCs exposed to doxycycline for 2 days showed that the protein was expressed in an inducible manner. (C) Immunocytochemistry of iPSC neurons (day 60 of differentiation) showing expression of the pan-neuronal filament markers tau and ßIII-tubulin and the synaptic protein synapsin-I in most cells. A subpopulation of GABA positive cells was present as well as a few cells positive for the neuroprogenitor markers nestin. Scale bars. 200 µm. Representative images from three independent neuronal differentiations. (D) Western Blot analysis for tau, ßIII-tubulin and synapsin-I expression in undifferentiated iPSCs (day 0) and iPSC neurons (day 59 of differentiation). Representative blots of three independent neuronal differentiations. (E) RT-qPCR for gene expression of pluripotency marker POU5F1 (Oct-4), pan-neuronal markers TUBB3 (ßIII-tubulin), MAPT (tau), MAP2 (microtubule associated protein-2) and RBFOX3 (NeuN), glutamatergic markers CAMK2A, SLC17A7, SLC17A6 and GABAergic markers GAD1, SLC6A1, SLC6A11 and SLC32A1 in iPSC neurons (day 60 of differentiation) versus undifferentiated iPSCs (day 0). Data are mean±SEM (n = 3, m = 4) and analyzed by Students unpaired t-test, ns. non-significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. (F) Changes in intracellular calcium in iPSC neurons (day 52/53 of differentiation) following application of the neurotransmitters, glutamate (300 µM glutamate +10 µM glycine), NMDA (40 µM) and GABA (100 µM), or depolarization of the membrane potential by addition of extracellular potassium chloride (25 mM). Representative traces are shown. (G) Spontaneous calcium oscillations in iPSC neurons (day 59/60 of differentiation) were eliminated by addition (timepoint marked with triangle) of 1 µM tetrodotoxin (grey traces) but not buffer (black traces). Representative traces are shown.

Fig 3: Melatonin promotes the neuronal differentiation of N2a cells. (A) The N2a cells gross morphology with or without 10 nmol/L melatonin treatment was presented (left). Magnification, 100×; scale bar, 10 µm. Neurite extension length was measured in six random microscopic fields after 0, 0.1, 1, or 10 nmol/L melatonin treatment. (B) Melatonin-treated cells were assessed for HAS1, HAS2, HAS3, GFAP, TUBB3, LC3, and GAPDH expression using Western blot analysis. GFAP and TUBB3 were selected as differentiation markers. (C) Differentiated N2a cells were identified via immunofluorescence staining of GFAP-, TUBB3-rhodamine, and HAS3-FITC after treatment with 0 or 0.1 nmol/L melatonin. Magnification, 630×; scale bar, 25 µm. The data are presented as the mean ± SD; *P < 0.05 compared with the control group

Fig 4: Melatonin inhibits tumor growth and promotes autophagy in vivo. (A) Tumor size was reduced by melatonin treatment. PBS, 40 mg/kg melatonin or 80 mg/kg melatonin was intraperitoneally injected into nude mice once a day for 3 weeks. The horizontal bars indicate the average tumor size. (B) Tumor sections from each group were analyzed for HAS3, GFAP, TUBB3, cytochrome C, and LC3 via IHC staining. (C) Each group of tumor lysates was assessed for HAS3, TUBB3, GFAP, LC3, and GAPDH. The results were normalized to GAPDH. (D) Autophagosomes were detected by TEM in tumor tissue from each group. Magnification, 12 K; scale bar, 1 µm. The data are presented as the mean ± SD; *P < 0.05

Fig 5: Overexpressing HAS3 in the N2a cells significantly promotes cell differentiation. (A) HAS3 overexpression plasmid was transfected into the N2a cells. The protein level of HAS3 and differentiation markers (TUBB3 and GFAP) were detected by Western blot assay (left panel). Differentiated N2a cells were calculated based on 1000 random cells in the vector control group and the HAS3 overexpression group. The morphological change after HAS3 overexpression was captured by microscopy. Magnification, 100×; scale bar, 10 µm. (B) Neurite length was measured in six random microscopic fields for both the vector control group and the HAS3 overexpression group at 24, 48 and 72 h. (C) Differentiated N2a cells were identified by immunofluorescence staining after transfection with an HAS3 expression plasmid. TUBB3-rhodamine was used as a marker for differentiated N2a cells. Endogenous and overexpressed HAS3 were identified via HAS3-FITC staining. Magnification, 630×; scale bar, 50 µm. The data are presented as the mean ± SD; *P < 0.05 and **P < 0.01