Gentamycin

First, E. coli K12 (MG1655) was been sequenced (Shanghai Majorbio Bio-pharm Technology Co., Ltd), which was designated as the original wild type strain (Table 1). Activating E. coli from storage tube with glycerol stock which stored in − 80 °C, expanding propagating on a Luria Bertani (LB) agar plates, cultured at 37 °C for 16 h. The selected seed strain was cultured in LB broth at 37 °C for 12 h for following selected experiments (Li et al. 2016 (link)).Table 1

The MIC of each antibiotic to wild-type E. coli K12

No	Antibiotics	Abbreviation	Classification	Stock solution (mg/L)	MIC (mg/L)
1	ciprofloxacin	Cip	Quinolones	200	0.2
2	tetracycline	Tet	tetracyclines	10	2.34
3	Gentamicin	Gen	Aminoglycosides	10	8.75
4	polymyxin B	Pol	Polypeptides	10	0.94
5	erythromycin	Ery	Macrolides	6.4	15
6	chloramphenicol	Chl	chloramphenicols	30	4.69

The involved antibiotics: chloramphenicol (Chl), ciprofloxacin (Cip), erythromycin (Ery), gentamycin (Gen), tetracycline (Tet), and polymyxin B (Pol) and cupric (CuSO4·5H2O) were get from Solarbio, Inc. (Shanghai, China). 90% inhibition of growth was regarded as the MIC of each antibiotic, which was determined as Additional file 1: Test S1 described, and the MIC of copper ions was also investigated with the same method. The detailed accounts are displayed in Additional file 1: Text S1. The tetracycline resistant cultures were kept from light so as not to degrade the antibiotic.

Cells (8 × 10³ cells per well) suspended in complete growth medium (RPMI1640) supplemented with 10% FBS and 1% gentamycin (Solarbio) were added to 0.3% agar with or without various concentrations of Ipriflavone in a top layer over a base layer of 0.6% agar containing the same concentration of Ipriflavone as the top layer. The cultures were maintained at 37°C in a 5% CO₂ incubator for 2 weeks. Colonies were visualized using an inverted microscope and quantified using the Image‐Pro Plus software (v.6) program (Media Cybernetics,Rockville, MD, USA).

Primary cortical neurons were prepared from E16-E18 embryos of Kunming mice. Specifically, embryonic cortices were dissected in cold DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and then digested using 2 mg/ml papain (CHI). The resulting cell suspension was centrifuged at 1500 rpm for 5 min. The cells were gently resuspended in DMEM supplemented with 10% FBS and gentamycin (50 μg/ml, Solarbio) and filtered through a 70-μm cell strainer (Corning Falcon). Then, the cells were seeded at a density of 1.2 × 10⁶ cells/ml in culture dishes coated with 20 μg/ml poly-D-lysine (Sigma). The neurons were cultured at 37 °C in a humidified incubator with 5% CO₂/95% air, and the medium was replaced 6 h afterwards with neurobasal medium (Gibco) supplemented with B27 supplements (Gibco) and L-glutamine (Gibco). After 6 days in vitro (DIV 6), the neural cultures were subjected to hypoxia treatment and protein extraction. N2a cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai; Institution Code: CBTCCCAS) and were cultured at 37 °C in a humidified incubator with 5% CO₂/95% air with MEM (Gibco) supplemented with 1.5 mg/ml NaHCO₃, 0.11 mg/ml sodium pyruvate (Gibco), and 10% FBS.