Pi3k inhibitor ly294002

The human non-small cell lung cancer cell line A549, HCC827 and H1975 were obtained from American Type Culture Collection (MD, USA). And human normal cell line of MRC-5, L02 and HK2 were purchased from the KeyGEN BioTECH (Nanjing, China). The human NSCLC cell lines A549, HCC827 and H1975 as well as L02 cells were cultured in RPMI 1640 (HyClone, UT, USA) containing 10% (v/v) fetal bovine serum (HyClone), and 1% penicillin/stretomycin (Invitrogen, CA, USA). MRC-5 and HK2 cells were grown in DMEM medium supplemented with (v/v) 10% heat-inactivated FBS and 1% penicillin/stretomycin (Invitrogen). Then, all cells were cultured at 37°C in a humidified atmosphere of 5% CO₂. Juglanin (CAS: 5041-67-8, HPLC≥98%), isolated from leaves of English walnut (Juglans regia), was purchased from Shanghai ZeYe Biological Technology Co., LTD. (Shnghai, China). P53 inhibitor Pifithrin-α (PFT-α, Sigma-Aldrich, MO, USA), NF-κB inhibitor pyrollidine dithiocarbamate (PDTC, Sigma-Aldrich), PI3K inhibitor LY294002 (Selleck Chemicals, TEX, USA), p38 inhibitor p79350 (Beyotime, Jiangsu, China), ERK1/2 inhibitor PD98059 (Selleck Chemicals) and c-Jun N-terminal kinases (JNK) activator Anisomycin (Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (DMSO, KeyGEN BioTECH) and freshly prepared for use.

Antibodies against RUNX1, SLUG and N-cadherin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against RUNX1 for IHC were from Abcam (Cambridge, MA, USA). Antibodies against SNAI1, α-SMA, Vimentin, SMAD4, p110δ, p-AKT, p-p38, p-ERK and p-SMAD3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against GAPDH, and secondary HRP-conjugated goat anti-mouse and anti-rabbit IgG were purchased from Beyotime Biotechnology (Shanghai, China). Electrochemiluminescent (ECL) reagents were purchased from Thermo Fisher Scientific (San Jose, CA, USA). Recombinant human TGF-β was purchased from PeproTech (Rocky Hill, NJ, USA). P110δ inhibitor CAL-101, PI3K inhibitor LY294002 and SMAD3 inhibitor SIS3 were purchased from Selleck Chemicals (Houston, TX, USA). Folic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). The siGENOME SMARTpool human RUNX1 siRNA was obtained from Dharmacon (Lafayette, CO, USA). SMAD3, PTEN, ATP1B1, and PIK3CD siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine RNAiMAX and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The Dual-Glo Luciferase Assay System was purchased from Promega (Madison, WI, USA). The RNAiso reagent was obtained from TaKaRa Ltd. (Kyoto, Japan).