Trichostatin a tsa

Delphinidin chloride was purchased from Extrasynthèse (Genay, France) and was used at 10⁻² g/L. This concentration has been described to induce the maximal relaxing effect on ex vivo rat aorta^{13 (link)}, to prevent angiogenesis through an inhibition of migration and proliferation^{16 (link), 17 (link)} and to inhibit endothelial apoptosis^{51 (link)}. Delphinidin was diluted in dimethylsulfoxide (DMSO) from Sigma Aldrich (St Louis, MO). The final concentration of DMSO in experiments never exceeded 0.1%. Anti-CD3 (clone OKT3) and anti-CD28 (clone CD28.2) human antibodies were purchased from BioLegend® (San Diego, CA). Histopaque®1077, Histopaque®1083, thapsigargin, Phytohemagglutinin (PHA), phorbol-12-myristate-13-acetate (PMA), ionomycin, fulvestrant, and SKF96365 were purchased from Sigma-Aldrich. Mibefradil hydrochloride was purchased from Abcam (Cambridge, UK) and trichostatin A (TSA) from Santa Cruz Biotechnology (Santa Cruz, CA). U0126 was obtained from Calbiochem (San Diego, CA). RPMI-1640, Na-pyruvate, non-essential amino-acid (NEAA) and penicillin/streptomycin were purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) and Fluo-4 acetoxymethyl ester (AM) were purchased from Life Technologies (Carlsbad, CA).

Thirteen NSCLC cell lines, including eight ADC (H1299, H2030, H23, A549, H322, H1650, H1975, and H2228) and three SCC (H2170, H226, and H157) lines, together with two SCLC cell lines (COLO677 and H82), were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig, Germany). The cell lines were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Hamburg, Germany). For the transfected cells, 400 ng/µL of Geneticin (G418) (Santa Cruz Biotechnology, Dallas, TX, USA) was added into the cell culture medium. Normal human bronchial epithelial cells (HBEC) were obtained from Lonza (Cologne, Germany) and grown in BEG media (Cologne, Germany). Cells were maintained in a humidified incubator with 5% CO₂ at 37 °C.
For drug treatment, 11 lung cancer cell lines were seeded in six-well plates. When the cells reached 50% confluence, they were treated with a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-Aza) and a histone deacetylase inhibitor, Trichostatin A (TSA) (Santa Cruz Biotechnology, Dallas, TX, USA), respectively, as previously described [49 (link)].