(A) MYC levels were determined in D425 (primary) and D458 (metastatic) tumor cells treated with triptolide for 16 hours. Representative immunoblots are shown. (B) Similar cells were exposed to triptolide for 48 hours and cell viability was determined by MTT reduction. Nonlinear regression analyses on the mean ± SEM of data normalized to DMSO were performed (n = 3). (C) Wound healing assays were performed in HD:MB03 cultures treated with 10 nM triptolide. A wound healing ratio was calculated relative to initial measurement. Mean ± SEM of the wound ratio per day (n = 3) and representative images (scale bars: 400 μm) are shown. (D) mG3-2929 sphere cultures exposed to 10 nM triptolide were plated on poly-D-lysine–coated wells for 24 hours. The ratio of viable attached cells, as determined by MTT reduction, to total viable cells was calculated. Mean ± SEM of data normalized to DMSO was plotted (n = 3). Representative images (scale bars: 50 μm) are shown. (E) mG3-2929 cells were implanted into mice 15 days before starting vehicle or triptolide (0.4 mg/kg, i.p., daily) dosing for 5 days. Metastatic lesions outside of the posterior fossa were quantified in ×2.5-magnified H&E-stained brains (vehicle n = 4, triptolide n = 5). Detail of metastatic lesions in representative images is shown. Arrows indicate tumor presence. (F) Mice harboring mG3-2929 tumors were similarly treated with vehicle or triptolide for 5 days, before quantifying numbers of positive cells in metastatic regions by IHC analyses (MYC n = 4, Ki67/C-Casp3 n = 3). Representative images (scale bars: 50 μm) are shown. In all cases, brains were harvested 6 hours after the last injection. Unless otherwise indicated, all results are presented as mean ± SEM of data normalized to 1 vehicle-treated animal. Statistical significance was assessed using an unpaired, 1-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.