Bcl 2

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 cell lysate was prepared with RIPA (Radioimmunoprecipitation assay) buffer and the total protein estimation was measured using Pierce BCA protein assay kit (Thermo Fisher Scientific, USA) as per the manufacturer’s instructions. The protein resolved in 10% SDS-PAGE was transferred onto Hybond-P PVDF membrane (Amersham Pharmacia Biotech, UK), blocked with 5% BSA (Company, country) and probed with primary antibodies for pNF-κB, NF-κB (Cell Signaling Technology, USA), Bcl-2 (Thermo Fisher Scientific, USA), Bax and β-actin (Santa Cruz Biotechnology, USA). Then the blots were probed with corresponding species-specific HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK) and the images were captured using FluorChem FC3 gel documentation system (Protein Simple, California, USA). The intensity of the bands were assessed using Image-J software (NIH, Bethesda, USA). β-actin was used as loading control.

Protein levels were assessed by Western blotting. Antibodies used to detect the following proteins are shown as catalogue numbers in parentheses: Bax (33-6400, Thermo Fisher Scientific Ltd., Waltham, MA); Bcl-2 (MA5-11757, Thermo Fisher Scientific Ltd., Waltham, MA); caspase-3 (MA1-91637, Thermo Fisher Scientific Ltd., Waltham, MA); p-Akt (9271, Cell Signaling Technology, Shanghai, China); Akt (9272, Cell Signaling Technology, Shanghai, China); and PI3K (4292, Cell Signaling Technology, Shanghai, China). A bicinchoninic acid assay kit (7780, Cell Signaling Technology, Shanghai, China) was used to measure protein concentrations prior to gel electrophoresis. Proteins were separated via 10% (w/v) sodium dodecyl sulphate–polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes; the membranes were blocked with 5% (w/v) blocking solution (non-fat milk) and incubated in blocking buffer with the primary antibodies overnight at 4 °C. Goat secondary antibodies conjugated with horseradish peroxidase were added, and a chemiluminescence kit was used to detect proteins.

Given that, following the cell viability test, the most affected cell line was HT-29, it was decided that the influence of 5FU, PBT, and PBT-5FU on gene expression should be established by applying the RT-PCR method [15 (link)] to this cell line. To evaluate the expression of the Bax, Bcl-2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and Bad (Eurogentec, Seraing, Belgium), the cells (1,000,000 cells/well) were cultured in 6-well plates. After reaching a confluence of approximately 80%, the cells were stimulated with test samples for a period of 72 h. After this time, RNA was isolated using Trizol reagent and the Quick-RNA™ purification kit, and its amount was determined using a DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA). Finally, RNA transcription was completed using the Maxima^® First Strand cDNA Synthesis Kit, and quantitative real-time PCR analysis was performed using the Quant Studio 5 real-time PCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in the presence of Power SYBR-Green PCR Master Mix.

After completing the mitral valve procedure, a left atrial tissue sample was
taken just before cross-clamp removal and the sample was stored in
paraformaldehyde at 4°C. Immunohistochemistry was performed (formalin/PFA-fixed
paraffin-embedded sections) using the avidin-biotin-peroxidase method (Zymed,
San Francisco, CA). Sections were incubated in iNOS (ready-to-use, prediluted.
Thermo Scientific, UK) Bcl-2 (ready-to-use, prediluted. Novocastra Laboratories,
UK), VEGF (NeoMarkers, 1:50), annexin (Zeta, 1:100) and eNOS (Anti-eNOS
antibody, 200 µl, Abcam) for 24h at 4°C in a humidified chamber.
Aminoethyl carbazole (AEC) is the chromogen of choice when performing
immunoperoxidase staining for 5 minutes at room temperature. After
counterstaining with Mayer's hematoxylin, immunoreactivity was examined using a
light microscope (Leica DM6000 B; Leica Microsystems Inc.; Buffalo Grove,
Ill).
In each group, the intensity of the positive immune stained cells in each section
was assessed by visual observation. Immunoreactivity was then graded according
to a 4-degree semiquantitative scale: minimal immunostaining (+), mild
immunostaining (++), moderate immunostaining (+++), or severe immunostaining
(++++).

A RT-PCR analysis was conducted in order to establish the influence of Met and Cet on gene expression. To determine the effects of the samples on the Bad, Bax, Bcl-2, and Bcl-xL genes, human keratinocytes were cultured in 6-well plates at 10⁶ cells/well. After reaching a 90% confluence, the cells were stimulated for a period of 24 h with Met and Cet in a concentration of 150 µg/mL. Total RNA was isolated from HaCaT cells using Trizol (cat. no. 15596026) purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and Quick-RNA ™ (cat. no. R1054) purification kit (Zymo Research) was used for RNA purification. The next step of the procedure was to obtain cDNA by reverse transcription reaction using Maxima^® First Strand cDNA Synthesis Kit (Fermentas, cat no. K1641). Quantitative real-time PCR analysis was performed using the Quant Studio 5 real-time PCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The analyzed mixture consisted of 20 µL solution and contained the following: i) Power SYBR-Green PCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA, cat. no. 4309155), cDNA samples, the sense and antisense primer and pure water. The primers used are shown in Table 1 and included: 18S (housekeeping genes), Bax, Bcl-2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), Bad (Eurogentec, Seraing, Belgium), and Bcl-xL (Eurogentec, Seraing, Belgium).