Phosphate buffered saline (pbs)

The four cell lines of human breast carcinoma utilized in this study were acquired from ATCC, Manassas, USA. Cell viability assays (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet)) were bought from Sigma-Aldrich, Taufkirchen, Germany. Multi-well plates, culture flasks and additional plastics for cell culturing were bought from TPP, Trasadingen, Switzerland and Greiner Bio-One GmbH, Frickenhausen, Germany. Fetal calf serum (FCS), RPMI cell culturing medium, L-glutamine, trypsin/ethylenediaminetetraacetic acid (EDTA) and phosphate-buffered saline (PBS) were all obtained from Capricorn Scientific GmbH, Ebsdorfergrund, Germany. The reagents for fluorescence-activated cell sorting (FACS) were obtained from several suppliers: Thermo Fisher Scientific: annexin V/propidium iodide (AnnV/PI) and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM); BD horizon: dihydrorhodamine (DHR) and carboxyfluorescein succinimidyl (CFSE); Sigma Aldrich: acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI); R & D scientific: ApoStat.

Torreya nucifera seed oil (TNSO) was obtained from Durae Corporation (Gunpo, Korea). Bovine serum (BS) was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Hyclone (Logan, UT, USA). Penicillin, streptomycin and phosphate-buffered saline (PBS) were purchased from Capricorn (Ebsdorfergrund, Hesse, Germany). Dimethyl sulfoxide (DMSO), insulin, dexamethasone (DEX), 3-iso-butyl-1-methylxanthine (IBMX), 3,3′,5-Triiodo-L-thyronine (T3) and the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

For separation of cytosol and nucleus protein fraction, differentiated THP-1 cells were washed and scraped into PBS (Capricorn Scientific GmbH, Ebsdorfergrund, Germany). After centrifugation (250 ×g, 4°C, 2 min) cells were lysed in Buffer 1 (10 mM Hepes pH 7.5, 10 mM KCL, 0.1 mM EDTA, 0,1 mM EGTA, 1× Protease inhibitor (Complete Mini Protease inhibitor Cocktail, Roche, Germany), 0.5 mM DTT) and incubated at 4°C on ice (15 min). Lysed cells were drawn 7–8 times through a 26 G needle and centrifuged (4,600 ×g, 4°C, 2 min). Cytosolic supernatant was centrifuged (20,000 ×g, 20 min, 4°C) and was used for Western Blot. Nucleic pellets were washed two times with Buffer 1 and were lysed with Buffer 2 (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1× Protease inhibitor (Roche), and 0.5 mM DTT) on a shaking incubator at 4°C for 1 h. Nucleic fractions were centrifuged (14,000 rpm, 4°C, 20 min) and supernatant was taken for Western Blot.

Ham’s F12 medium was obtained from GE Healthcare Europe (Freiburg, Germany). RPMI-1640, DMEM, GlutaMAX, Penicillin/Streptomycin and FCS were purchased from Life Technologies (Darmstadt, Germany). PBS was acquired from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Opti-MEM was obtained from Thermo Fisher Scientific (Frankfurt, Germany). Phorbol 12-myristate 13-acetate (PMA) was supplied by Sigma-Aldrich Chemie (Munich, Germany). LPS (Salmonella minnesota R595, TLR grade) was obtained from Enzo Life Sciences (Lausen, Switzerland). Pam3CSK4 was purchased from Invivogen (San Diego, USA). JAK inhibitor Ruxolitinib was obtained from Biozol Diagnostics Vertrieb GmbH (Eching, Germany). Polymyxin B was purchased from Merck Millipore (Billerica, USA). Antibodies were obtained from Abcam (Cambridge, UK): Mx1 (ab95926), influenza nucleoprotein (9G8); Cell Signaling (Cambridge, UK): phospho-IRF-3 (Ser396)(4D4G), phospho-TBK-1 (Ser172)(D52C2), TBK-1 (61223S), phospho-STAT1 (Tyr)(58D6), STAT1 (D1K9Y), mouse anti rabbit (L27A9), IRAK-1 (4359S), phospho-p38 (Thr180/Tyr182)(9211S), p38 (9212S); Thermo Fisher Scientific: goat anti-mouse (alexa fluor 488); ProteinTech: anti-IRF-3 (66670-1) or Santa Cruz Biotechnology (Heidelberg, Germany): β-actin (C4), anti-mouse (mIgGκBP-HRP, sc-516102). All other applied chemicals were of analytical grade and acquired from commercial sources.

The investigated cell lines, PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) were purchased from ATCC (Manassas, VA, USA). The cell culture medium RPMI 1640, the supplements FCS and l-glutamine, as well as PBS and trypsin/EDTA were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Culture flasks, multi-well plates, and further cell culture plastics were purchased from Greiner Bio-One GmbH (Frickenhausen, Germany) and TPP (Trasadingen, Switzerland), respectively. Anti-proliferative and cytotoxic effects, respectively, of the compounds, were investigated by performing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet)-based cell viability assays (Sigma-Aldrich, Taufkirchen, Germany), respectively.

The HepG2 (5 × 10⁵) cells were seeded onto 6-well cell culture plates and incubated for 24 h in a fully supplemented EMEM (Corning®) with the addition of the analysed compounds at a concentration corresponding to the total IC₅₀ values (34–5 µM, 35–4.5 µM, 36–4 µM, 37–4.5 µM). The DNA content was determined using flow cytometry with PI (Sigma-Aldrich, Steinheim, Germany) staining. After incubation, the cells were trypsinized and washed twice with 1 ml of PBS (Capricorn). In the next step, the cells were fixed with an ice-cold 80% ethanol. After incubation for 1 h at 4 °C, the fixed cells were washed twice with PBS and suspended in the staining PI/RNAse A/PBS buffer (PI 50 µg/ml, RNAse A 100 µg/ml) for 30 min at 37 °C in the dark. PI (Sigma) fluorescence was measured using a FACSCalibur (Becton Dickinson), and data were analysed using the FlowJo software (Becton Dickinson).