Caspase 8

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.

The total RNA content was extracted using peqGold RNAPureTM Package (Peqlab Biotechnology GmbH, Erlangen, Germany) according to the manufacturer’s instructions, and a DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA) was used to measure the total concentration of RNA. Reverse transcription was performed using the Maxima^® First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the samples were incubated into Tadvanced Biometra Product line (Analytik Jena AG, Göttingen, Germany) according to the following thermal program: 10 min at 25 °C, 15 min at 50 °C and 5 min at 85 °C. The quantitative real-time PCR was conducted using a Quant Studio 5 real-time PCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The analysis was performed using 20 µL reactions containing Power SYBR-Green PCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA), samples’ cDNA, the sense and antisense primer and pure water. The following primer pairs were used (Table 2): 18S and GAPDH (as housekeeping genes), Bax, Bcl-2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), Bid, Bad, Bak, Bcl-xL, caspase 3, caspase 8 and Apaf-1 (Eurogentec, Seraing, Belgium). Normalized, relative expression data were calculated using the comparative threshold cycle (2-ΔΔCt) method.