5 bromo 3 deoxyuridine brdu

Anemoside B4 (B4, chemical structure C₅9H₉6O₂₆, molecular weight = 1,221.38, purity >98%) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products. PDGF-BB was purchased from PEPROTECH. The small molecule inhibitors LY294002 (PI3K/Akt inhibitor), SD98059 (ERK inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies against Akt/phospho-Akt, ERK/phospho-ERK, p38 MAPK/ phospho-p38 MAPK, and JNK/phospho-JNK were from Cell Signaling Technology (Massachusetts, USA). Antibodies against α-SMA, Calponin, SM22-α, and BrdU were obtained from Abcam (Cambridge, UK). TUNEL staining kit was obtained from Roche. 5-Bromo-3′-deoxyuridine (BrdU) was purchased from Sigma-Aldrich (St. Louis, MO). See Supplementary Tables 1, 2 for detailed information.

Primary cortical neuron culture was performed as described [13 (link)]. The primary cortical brains were collected from E15.5 mouse embryos and dissociated to single cells after digestion with trypsin (Celgene, Summit, NJ, USA). 1 × 10⁵ neuronal cells were plated in poly-D-lysine coated 24-well plates and cultured in Neurobasal medium/DMEM (1 : 1) with B27 supplement, penicillin, streptomycin, and glutamine (Gibco-BRL, Gaithersburg, MD, USA) at 37°C in a humidified incubator with 5% CO₂/95% air. The day of plating was considered day in vitro 0 (DIV 0). On DIV 1, cells were received OP at a low concentration 10⁻⁶ M and 10⁻⁸ M. On DIV 4, neurons were harvested for quantification of axon and dendrite morphology. For proliferative experiments, neurons were incubated with 5-bromo-3-deoxyuridine (BrdU) (10 mM/mL, Sigma-Aldrich, St. Louis, MO, USA) after 12 h treated with OP. Then, neurons were harvest after 2 h BrdU-treatment.