Ml385

Three-week old gels were treated with either 10 nM vincristine (VCR) (S1241; Selleckchem), 5 µM NRF2 inhibitor ML385 (6243, Bio-Techne) or both for one week. DMSO was used as a vehicle control. Drugs were renewed 24, 72 and 144 h after the first dose. In order to assess the cell’s recovery potential all gels were washed and covered with fresh, drug-free medium one week after the final VCR treatment and monitored for a further four weeks (total experiment time: 8 weeks). For cell viability assays, Prestoblue reagent (Thermo Fisher) was used according to the manufacturer’s instructions and fluorescence intensity was measured after 40 min incubation time using a microplate reader (FLOUstar Omega; BMG Labtech, Ortenberg, Germany). All gels were washed four times with HBSS buffer (Thermo Fisher) before fresh medium was added to each well. Cell viability within each gel was measured weekly after drug or vehicle treatment.

MIO-M1 cells were treated with different concentrations of recombinant human VEGF (Sigma-Aldrich, St. Louis, MO, USA), of H₂O₂ (Sigma-Aldrich), or with 0.1 µM Apatinib (Selleck Chemicals, Houston, TX, USA), a VEGFR2 inhibitor. Before the treatments, the growth medium was replaced with FBS-free medium. H₂O₂- and VEGF-treated cultures were also treated with 5 µM ML385 (Bio-Techne, Minneapolis, MN, USA), an inhibitor of Nrf2, and with 5 µM acriflavine (ACF, Sigma-Aldrich), a HIF-1 inhibitor. All treatments were performed for 24 h.

N-Acetyl cysteine (NAC, Beyotime Biotechnology, China) was diluted in PBS and daily administered through intraperitoneal injection (200 mg/kg, i.p.) 30 min ahead of behavior test. The dosage of NAC is based upon previous literatures [23 (link), 24 (link)]. H₂O₂ was diluted in PBS and injected (100 nmol/50 μl) to dorsal region of hindpaw. The control group of rats was injected with vehicle (PBS) only. ML385 (Tocris, USA) was dissolved in DMSO and further diluted in PBS for local hindpaw injection (400 μg/site in 50 μl volume). ML385 was injected 30 min before each EA intervention.