Mccoy s 5a

HEK293T (ATCC, CRL-3216, human embryonic kidney) and A549 (ATCC, CCL-185 human lung adenocarcinoma) cells were cultured in DMEM high glucose (Invitrogen, Darmstadt, Germany), BxPC3 (DSMZ, ACC-760, human pancreatic adenocarcinoma) cells in RPMI 1640 (Invitrogen) and HCT116 (ATCC, CCL-247, colorectal carcinoma) cells in McCoy’s 5A (Pan-Biotech, Aidenbach, Germany) supplemented each with 10% tetracycline-free FBS and antibiotics. Transient transfections were performed using Viafect (Promega, Mannheim, Germany). Production of lentiviral particles were done in HEK293T cells as described previously (Kranz et al., 2017). For transduction, 2 × 10⁵ cells were incubated with 2 × 10⁶ lentiviral particles in the presence of 8 µg/ml polybrene for 24 h followed by selection with 2 µg/ml puromycin for 5 days. All experiments were performed with mycoplasma-free cells.

The analyzed compounds (aspirin and 5-fluorouracil) and the other reagents used in the present study dimethyl sulfoxide (DMSO), fetal calf serum (FCS), penicillin/streptomycin, trypsin-EDTA solution, phosphate saline buffer (PBS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 4′,6-Diamidino-2-phenylindole dihydrochloride, and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), were purchased from Sigma Aldrich, Merck KgaA (Darmstadt, Germany), and Alexa Fluor^® 555 Phalloidin was acquired from Cell Signaling USA.
Cell lines were cultured in the specific media DMEM (P04-03550) and McCoy’s 5A (P04-05500), which were purchased from PAN Biotech GmbH (Aidenbach, Germany). As part of RT-PCR, the following primers were used: 18S, Bax, Bcl-2 purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and Bad, caspase 3, and caspase 8, purchased from Eurogentec (Seraing, Belgium). All reagents presented appropriate characteristics for use in cell culture.

HEK293 (CRL-1573), MIA PaCa-2 (CRL-1429), HT-29 (HTB-38), PC3 (CRL-1435), LNCaP (CRL-1740), and PNT1a (CRL-11609) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Each cell line was grown in MEM, DMEM, McCoy’s 5A, and RPMI medium (PAN Biotech, Aidenbach, Germany) completed by 10% fetal bovine serum (Gibco, Paisley, PA4, United Kingdom), 10000 U/mL penicillin, and 10 mg/mL streptomycin (PAN Biotech, Aidenbach, Germany) at 37°C in 5% CO₂ incubator (HERAcell 150, Thermo Scientific (Paisley, PA4, United Kingdom), respectively. Anti-His tag (1:1000), anti-GAPDH (1:1000) primary antibodies, HRP-conjugated anti-rabbit secondary antibody (1:3000), and HRP-conjugated anti-goat secondary antibody (1:3000) were obtained from Cell Signaling Technologies (Danvers, MA, USA). Anti-GHRH primary antibody (1:500) and anti-GHRHR antibody (1:1000) were purchased from Origene (Rockville, USA) and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively. His-tagged (pCMV3-SP-N-His-NCV; CV023) and His-tagged GHRH (pCMV3-SP-His-ORF His-GHRH) plasmids were purchased from Sino Biological (Wayne, PA, USA).