Fig 2: 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 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: Cochlear neuronal development in E70 and E77. (A-E) Cochleovestibular ganglion in E70 cochlea. Expression of TUBB3, ISL1, and NEUROD1 was observed at E70 (A). Expression of POU4F1, SOX2, and PRPH was also observed in E70 cochleovestibular ganglion (B, C, and D). However, expression of MAFB and GATA3 was not detected in E70 cochleovestibular ganglion (D and E). (F-L) Spiral ganglion in E77 cochlea. Expression of TUBB3 and PRPH was observed in E77 spiral ganglion neurons (F). In E77, S100B-positive glial cells surrounding spiral ganglion neurons were observed (F). In E77, expression of NEUROD1 was observed in only an apical turn (asterisk in G), while RBFOX3 expression was observed in basal turns (H). No RBFOX3 expression was observed in the NEUROD1-positive spiral ganglion neurons (I). Expression of GATA3, ISL1, and MAFB was observed in the spiral ganglion neurons in E77 (J-L). The nuclei were counterstained with Hoechst (blue). Scale bar: 50 µm in A-E, G, and I, 100 µm in F, H, and J-L

Fig 5: Starvation induces N2a cell differentiation via HAS3 upregulation. (A) N2a cells were treated with 0%-10% serum for 24 h. The treated cells were assessed for HAS1, HAS2, HAS3, TUBB3, GFAP, ß-tubulin, and a-actin by Western blot analysis (A = attached cells, D = detached cells). The results were normalized to a-actin. N2a cells were treated with 2.5 or 10% serum. Differentiated cells were stained with HAS3-rhodamine and ß-tubulin -FITC. The colocalization of HAS3 and ß-tubulin was measured by FRET analysis. Magnification, 630×; scale bar, 50 µm. (B) N2a cells were treated with 0%, 0.1%, 1% or 10% serum. The morphology of the differentiated cells was captured by microscopy. Magnification, 100×; scale bar, 10 µm. Differentiated cells were assessed for HAS3 and GAPDH by Western blot analysis. The results were normalized to GAPDH. Neurite length was measured in six randomly selected microscopic fields using ImageJ software. The data are presented as the mean ± SD; **P < 0.01 compared with the control group