Trypsin edta

The GT1-7 cell line (mouse GnRH neuronal cell line) was kindly provided by Prof. P. Mellon (University of California, San Diego, CA, United States). GT1-7 cells were grown in a monolayer culture in DMEM (Corning) supplemented with 10% FBS (Gibco), 1% penicillin-streptomycin (Macgene), and 0.5% Mycoplasma Prevention Reagent (Yeasen Biotech). The cultures were incubated at 37°C in an atmosphere of 5% CO₂ in a 25 mm flask (Corning) for 2 days after seeding, with a medium change at 24 h. The cells were then washed twice with PBS and digested with 0.25% trypsin-EDTA (MacGene). When more than half of the cells were observed to become round under a microscope, serum-containing medium was added to terminate the digestion. After obtaining a single-cell suspension, the cells were cultured in an incubator and inoculated three times for subsequent experiments. The cells were treated with different treatments in serum-free medium (SFM) for 24 h depending on the experiment.

Zinc nitrate hexahydrate (Zn(NO₃)₂ 6H₂O) was purchased from Sigma-Aldrich. 2-methylimidazole was purchased from Macklin. Trypsin-EDTA was obtained from MACGENE. PBS buffer and DMEM (high glucose) were purchased from Hyclone. BIBR 1532 was obtained from Aladdin and the structure of BIBR 1532 was shown in Figure S1. Fluorescein, free acid (green fluorescence signal) was purchased from Coolaber. 6-aminofluorescein (6-AF, green fluorescence signal), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Yuanye Biological. Hoechst 33,342 and Lyso-Tracker Red were purchased from Beyotime Biotechnology. Recombinant anti-lysosomal associated membrane protein 1 (LAMP1) antibody, recombinant anti-cathepsin B antibody and recombinant anti-GAPDH antibody were purchased from Abcam. Anti-rabbit IgG, HRP-conjugated antibody was purchased from Cell Signaling Technology. A549 cell line was obtained from American Type Culture Collection (ATCC).