Cryptotanshinone

Salivary gland mucoepidermoid carcinoma MC3 cells which were kindly provided by Professor Wu Junzheng in the Forth Military Medical University (Xi'an, China), were isolated by repeated in situ-transplants of MEC-1 human mucoepidermoid carcinoma cell line derived from palatal salivary gland as described in the study by Wen et al (16 (link)). YD15 cells were obtained from Professor Jin Kim in Yonsei University (Seoul, Korea), which was derived from salivary gland mucoepidermoid carcinoma of tongue origin (17 (link)). MC3 cells were grown in DMEM and YD15 cells were grown in RPMI-1640; both types of media were supplemented with 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin and streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. An equal number of cells were seeded and allowed to attach. All experiments were performed in cells cultured at 50–60% confluence. Methanol extract of Potentilla discolor (MEPD) was supplied from Korea Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (Cheongju, Korea), and cryptotanshinone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Each chemical was dissolved in dimethyl sulfoxide (DMSO), aliquoted, and stored at −20°C.

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Lots p997 and p1028) and cultured in endothelial basal medium (EBM; Lonza), supplemented with EGM SingleQuots (Lonza) and 10% foetal calf serum (FCS; Invitrogen. San Diego, CA, USA). Human coronary artery endothelial cells were purchased from Promocell and cultured in endothelial growth medium MV (Promocell). Primary cells were used between Passage 2 and 4 for experiments. HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% FCS, D-Glucose, Pyruvate, Penicillin/streptomycin, and minimum essential media non-essential amino acid mix (Sigma Aldrich); Hek293T cells were cultured in DMEM with 10% heat inactivated FCS, D-Glucose, Pyruvate, and Penicillin/streptomycin. Cells were cultured at 37°C with 5% CO₂. Cell numbers were determined with a Nucleocounter NC-2000 (Chemometec A/S). HUVECs were stimulated with 100 ng/mL IL-6 (Peprotech) and sIL-6Rα (Peprotech) each in EBM for 10 min (for pSTAT3 Y705 western blots) or 4 h before cell lysates were prepared. A 20 µM Cryptotanshinone (CPT; Sigma Aldrich) in Dimethyl sulfoxide (DMSO) was added to cell culture medium 1 h prior to addition of IL-6 and sIL-6Rα. Equal volumes of DMSO were used as controls for CPT experiments.