Tanshinone iia

The materials for cell culture were purchased from Gibco (Grand Island, NY, USA). MAPK inhibitor, including PD98059 for ERK1/2, SP600125 for JNK, SB203580 for p38, and LY294002 for Akt, and AP-1 inhibitor, i.e., Tanshinone IIA, were purchased from Sigma (St. Louis, MO). Rabbit antibodies against MSH2, phosphor-ERK1/2, ERK1/2, phosphor-Akt, Akt, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). The control-, MSH2-, and c-jun-siRNA were purchased from Thermo (Waltham, MA). All others chemicals were obtained from Sigma (St. Louis, MO).

All solvents and reagents were reagent grade; H₂O was distilled and deionised. Tanshinone IIA was purchased from Sigma (St. Louis, MO) and was dissolved in ethanol (100%). The two turmeric preparations contained the curcuminoids curcumin, desmethoxycurcumin, and bis-demethoxycurcumin as follows: (1) Naturex #140500 (Avignon, France); 95% curcuminoids (high-performance liquid chromatography, Supplementary Fig. S1D), (2) curcumin powder 95%, 89.88% curcuminoids and volatile oils of turmeric rhizome (Dolcas Biotech LLC; PCCA, Houston, TX). Both were dissolved in dimethylsulfoxide (DMSO) (Sigma; St. Louis, MO), prior to addition to cell cultures. Resveratrol (99.0%; Jiaherb) and EGCG (95.3%; Taiyo Green Power Co., Ltd) were obtained from PCCA (Houston, TX), and were dissolved in ethanol. All solutions were sonicated for 10 min. The source of the compounds for the gene expression studies was as follows: curcumin (≥98% curcuminoid content) (CAS number 458-37-7) (Thermo Fisher Scientific, NJ, USA; prepared by Acros Organics for Thermo Fisher; stored at room temperature under nitrogen), (−)-epicatechin gallate, and Resveratrol (Thermo Fisher).

Tissue culture plates, including 6- and 24-well ultralow attachment cluster plates, were obtained from Corning (Tewksbury, MA, USA). DHTS I, crytotanshinone, tanshinone I, and tanshinone II A were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell growth was assayed using a CellTiter 96® AQueous One Solution kit (Promega, Madison, WI, USA). The ALDEFLUOR™ Kit was obtained from STEMCELL Technologies Inc. (Vancouver, BC, Canada). Chemicals such as MβCD were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

Tanshinone IIA (T4952) and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (United States).; BPL (3-n-Butylphthalide, a marketed drug for acute ischemic stroke) (H20100041) was purchased from CSPC (China); BAY-11-7082 (a NF-κB inhibitor) (S2913) was purchased from Selleck (United States); ELISA Kit was purchased from R&D Systems (United States); superoxide dismutase (SOD) and malondialdehyde (MDA) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (China); total RNA Kit was purchased from Takara (Japan). Primary antibodies: NeuN (24307), Iba-1 (17198), iκB (76041), NF-κB (p65) (8242), GAPDH (5174) were purchased from Cell Signaling Technology (United States); p-iκB (ab133462) and p-NF-κB (p-p65) (ab239882) were purchased from Abcam (United States); CD16/32 (AF1460) and CD206 (AF2535) were purchased from R&D Systems (United States). Secondary antibodies: Alexa Fluor^® 488 AffiniPure Goat Anti-Rabbit IgG, Alexa Fluor^® 488 AffiniPure Donkey Anti-Goat IgG and Cy™3 AffiniPure Donkey Anti-Rabbit IgG were purchased from Jackson (United States).

The human NSCLC cell line A549 and PC9, and the Human Lung Fibroblast (HLF) cell line were supplied by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI1640 (Gibco, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) in a humidified incubator at 37 °C, 5% CO₂ atmosphere. Tanshinone IIA (Fig. 1a) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and prepared as a 10 mM stock solution in dimethylsulfoxide (DMSO) (St. Louis, MO, USA). The solution was serially diluted in a RPMI 1640 medium immediately prior to the experiments. ADM (Fig. 1b) was purchased from Sigma-Aldrich and prepared as a 10 mM stock solution in normal saline (NS) which was serially diluted in RPMI 1640 medium immediately prior to experiments. Pancreatin, penicillin and streptomycin were purchased from Gibco (Invitrogen Life Technologies, Carlsbad, CA, USA). All the reagents were of analytical grade.Fig. 1

The three-dimensional (3D) structure of  Tanshinone IIA (a) and ADM (b) (from PubChem compound http://pubchem.ncbi.nlm.nih.gov/)

Gamborgs B-5 basal medium, sucrose, 5-azacytidine and tanshinone IIA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tanshinone I, dihydrotanshinone I, and salvianolic acid B were purchased from Wuhan ChemFaces Biochemical Co., Ltd. (WUHAN, HUBEI, China). Rosmarinic acid was purchased from ChromaDex (Los Angeles, CA, USA). Cryptotanshinone was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). HPLC grade ethanol was purchased from Honeywell Chemicals (Seelze, Germany). HPLC grade acetonitrile was purchased from Fisher Scientific (Seoul, Korea).