Extraction and Analysis of Herbal Extract Compounds

SH003 was prepared as described in our previous study [26 (link)]. In brief, SH003 was provided from Hanpoong Pharm and Foods Company (Jeonju, Republic of Korea). Am, Ag, and TK were mixed at 1 : 1 : 1 ratio (w/w) and then extracted with 30% ethanol at 100°C for 3 h. Dried extracts were dissolved in 30% ethanol and stored at −80°C until use. DTX (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO and stored at −20°C.
Thiazolyl blue tetrazolium bromide (MTT) powder was purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated Annexin V apoptosis Detection Kit and 7-aminoactinomycin D (7-AAD) were purchased from BD Pharmingen™ (BD Biosciences, San Jose, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Anticleaved caspase-3, -PARP, -GAPDH, -p-EGFR (Tyr 1068), -p-EGFR (Tyr 1173), -EGFR, -p-AKT (Ser 473), -AKT, -p-C-Raf (Ser 338), -p-STAT3, and -STAT3 antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p-ERK and ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Ki67 and CD31 antibodies were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies for mouse and rabbit were purchased from SeraCare Life Sciences (Milford, MA, USA).