Actinomycin

FGF ligands (PeproTech) were used at 50 ng/ml for 4 h or overnight. c‐Jun N‐terminal kinase (JNK) inhibitor (SP600125, Sigma‐Aldrich, Milan, Italy) was used at 50 μM for 12 h. Bafilomycin A1 (Sigma‐Aldrich) was used at 100 nM for 3–4 h. Torin 1 (Cell Signaling) was used at 1 μM for 2 h. Concanamycin A (Sigma‐Aldrich, Milan, Italy) was used at 100 nM for 1 h. Proteasomal inhibitor (MG132, Sigma‐Aldrich, Milan, Italy) was used at 10 μM for 6 h. Actinomycin (Sigma‐Aldrich) was used at 1 μg/ml for 4 h. ER‐Tracker BODIPY Green (BODIPY™ FL Glibenclamide, for live‐cell imaging, Thermo Fisher) was used at 1 μM for 30 min at 37°C in DMEM (w/o supplements) in dark and then fixed with 4% PFA for 2 min at 37°C.

The experiments were conducted at a temperature of 25 °C. All experiments with intercalators and minor group binders were performed in TE buffer (Tris 10 mM, ethylenediaminetetraacetic acid 1 mM) pH 7.5, 100 mM NaCl, 0.01% NaN₃ and DMSO. Echinomycin was purchased from Merck and Actinomycin, DAPI and netropsin from Sigma-Aldrich. Thiocoraline was provided by Pharmamar. Concentrations used ranged from 100 nM to 10 μM. The different restriction enzymes were purchased from New England biolabs and used as 1:100 dilutions of the original stock: RsaI (10,000 U/ml, 50 nM dimer [1.7 μg/ml], MW: 19 kDa monomer), BspCNI (2000 U/ml, 2 μM [270 μg/ml], MW: 105 kDa), MnlI (5000 U/ml, 1.2 μM dimer [90 μg/ml], MW: 38 kDa monomer).

Following the standard procedure [17 (link)], caspase activion was detected in HUVEC cells incubated with NGR-hTNF or hTNF, in the presence of 1.5 µg/mL cycloheximide (Sigma-Aldrich), for 30 min. Then, the cells were washed and incubated in the presence of 1.5 µg/mL cycloexamide, without cytokines, for additional 4 h. After two washes with ice-cold PBS, the cells were lysed, and proteins resolved by SDS-PAGE. The activation of caspase 3 and 8 was detected by immunoblot, using the anti-caspase 3 and 8 (Cell Signaling Technology, Danvers, MA, USA), respectively. The detection of active caspase 3 on histological sections of CT26 tumor collected from mice treated with NGR-hTNF or hTNF (100 pg/mouse), and sacrificed after 24 h, was performed with the anti-Cleaved Caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA). For cytotoxicity analysis, based on protocols reported in the literature [72 (link)], subconfluent cells (HUVEC, MR300 or L-M) were incubated with the indicated cytokines concentrations for four hours, washed and then incubated with regular medium for additional 48 h, in the presence of 1 µg/ml actinomycin (Sigma-Aldrich), and 20 mM lithium chloride. Viability was evaluated by standard MTT colorimetric assay.

Monocytes isolated from healthy volunteers were exposed to 100 ng/ml LPS (Escherichia coli 0111:B4; Sigma, St. Louis, Missouri, USA) for 16 h and treated with 0.5 μg/ml STS (Enzo Life sciences, Farmingdale, New York, USA) for 8 h prior to harvest. Apoptosis was induced in transduced and control THP-1 cells with 0.5 μg/ml STS for 4 h in complete medium. To inhibit caspase activity, cells were pretreated with 100 μM Z-VAD-FMK (Sigma, St. Louis, Missouri, USA) for 1 h prior to STS treatment. To inhibit the mRNA and protein synthesis of Bak, cells were treated with 10 μg/ml actinomycin or 20 μg/ml CHX (Sigma, St. Louis, Missouri, USA), respectively, 1 h before STS stimulation.

Sulforaphane (≥95% HPLC), hemin, actinomycin, angiotensin II, N-acetylcysteine (NAC), dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and β-actin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). NG-nitro-L-arginine methyl ester (L-NAME), LY294002 (PI3K inhibitor), pyrazolopyrimidines (PP2, an Src kinase inhibitor) were bought from Calbiochem (La Jolla, CA, USA). Primary antibodies for eNOS (32027S), phospho-eNOS (Ser 1177) (9570S), Akt (4691s), phospho-Akt (Ser 473) (4060S), and phospho-Src (Tyr 416), (6943S) as well as horseradish peroxidase-conjugated anti-mouse (7076S) or anti-rabbit IgG (7074S) antibodies were obtained from Cell Signaling Technology (Beverly, MA). All other chemicals, unless otherwise noted, were pure, analytical grade. The cell culture media and reagents involved were obtained from Invitrogen (Carlsbad, CA, USA).