Pd98059

To determine the optimal concentration of the ERK_1/2 inhibitor PD98059 (ChemScene LLC, Monmouth Junction, NJ, USA), western blotting was performed on cells treated with PD98059 at three concentrations (10, 20, and 40 μM). The western blotting data were quantified by using Image Lab software (Version 5.0, BioRad, Hercules, CA, USA). After the optimal concentration was determined, cell proliferation and migration, and immunostaining for type I collagen were compared in response to treatment with shikonin with or without PD98059.

Cortical neurons were generated as described earlier. On day 8, the medium was totally removed and replaced with neuronal medium containing 5 μM CellEvent Caspase-3/7 Green Detection Reagent (Invitrogen, Thermo Fisher Scientific). Depending on the experiments, 5 μM z-VAD-FMK (Peptide Institute Inc., Osaka, Japan), 0.1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich), or different concentrations of PD98059 (ChemScene, Monmouth Junction, NJ) and SP600125 (ChemScene) were added to the medium. After replacing the medium, the plates were incubated in IncuCyte S3 (Sartorius, Goettingen, Germany) and imaged using the IncuCyte ZOOM imaging system (Essen BioScience, Sartorius) with images collected every 6 h from day 8 to day 15. During this period no medium exchange was performed. We defined neurite length per cell-body cluster area (mm/mm²) as neurite length (mm/mm²), and areas showing green fluorescence per total area (%) as caspase-3/7 positive area (%) according to the manufacturer’s instructions.