Emodin

Rats alveolar type II epithelial cell line RLE‐6TN (ATCC^® CRL‐2300™) and human alveolar epithelial cell line A549 (ATCC^® CCL‐185™) were obtained from ATCC (Manassas, VA, USA). RLE‐6TN cells were cultured in Ham's F12 medium with 2 mM L‐glutamine supplemented with 0.01 mg/ml bovine pituitary extract, 0.005 mg/ml insulin, 2.5 ng/ml insulin‐like growth factor, 0.00125 mg/ml transferrin, 2.5 ng/ml EGF (90%) and 10% FBS. A549 were cultured in F‐12K Medium with 10% FBS. Cells were cultured at 37℃ in 5% CO₂. The lung epithelial ell lines were authenticated using immunofluorescence staining (IF staining) with anti‐proSPC (ab90716; Abcam, Cambridge, UK).
Recombinant mouse NE protein (4517‐SE‐010) was obtained from R&D Systems (Minneapolis, MN, USA) and used to treat RLE‐6TN cells and A549 cells at concentrations of 50 nM, 100 nM or 200 nM. For Emodin treatment, Emodin (Sigma‐Aldrich, St. Louis, MI, USA) was firstly dissolved in DMSO (Sigma‐Aldrich) and further diluted to 10 µg/ml.¹⁶ The final DMSO concentration was less than 0.1% (v/v). Sivelestat (S7198; Sigma‐Aldrich), a neutrophil elastase inhibitor, was used to treat RLE‐6TN cells at a density of 100 µg/ml. The γ‐Secretase Inhibitors DAPT (ab120633, Abcam) was used as notch pathway inhibitor to RLE‐6TN cells at a density of 50 nM.

Cell culture materials were obtained from Invitrogen (Burlington, Ontario, Canada). The reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI) and the PureLink™ HiPure Plasmid DNA Purification Kit were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against PARP and Twist were purchased from GeneTex (Beverly, MA, USA). Anti-HER-2, anti-phospho-Akt (Ser473), anti-E-cadherin, anti-vimentin, anti-Snail, and anti-Slug antibodies were purchased from Cell Signaling (Beverly, MA, USA). Primary antibodies against ILK, phosphor-ILK (Thr173), phospho-mTOR (Ser2448), and phospho-GSK3β (Ser9) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against YB-1 and β-Actin were purchased from Millipore (Temecula, CA, USA). The antibody against HIF-1α was purchased from BD Biosciences Clontech (San Jose, CA, USA). For Western blotting, the secondary antibodies of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Millipore (Temecula, CA, USA), and enhanced chemiluminescence (ECL) reagents were purchased from Sigma-Aldrich. Emodin, AE, and rhein were purchased from Sigma-Aldrich. The RNAi Consortium of YBX-1 was selected by the National RNAi Core Facility.

GBM cells (1 × 10⁴) were plated in 96 well plates and treated with inhibitors of Calmodulin (CGS-9343B, Sigma), Casein kinase II subunit alpha (Emodin, Sigma), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (U73122, Sigma), Tyrosine-protein kinase ABL1 (Dasatinib, Santacruz) and B-cell lymphoma 2 (ABT199, Santacruz) for 24hr and subsequently incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT,100 μg/ml DMSO) in fresh culture medium for 3 hr. Optical density (OD) was taken at 550 nm with an ELISA reader (Thermo) as described elsewhere [105 (link)].

Reference substances for qualitative and quantitative analysis were trans-resveratrol (99% purity, Sigma-Aldrich, St. Louis, MO, USA), emodin (90% purity, Sigma-Aldrich), and polydatin CRS (trans-polydatin, 99.1% purity, EDQM—Council of Europe, Strasbourg, France). For extract preparation, deionized water and ethanol (AustrAlco, Spillern, Austria) were used. Samples of reference substances were prepared in p.a. grade methanol (VWR, Radnor, PA, USA). HPLC mobile phases were made up of ultrapure water (Barnstead Easypure RF, Barnstead) and HPLC-grade acetonitrile (VWR).