Bcl2 is classically described as a protein that provides survival advantages to cells (27,28)

Bcl2 is classically described as a protein that provides survival advantages to cells (27,28). cardiac cells. In addition, the results demonstrated a downregulation of prostaglandin E2 production VPS15 by the cardiac cells incubated with celecoxib, in a dose-specific manner. These results are consistent with the decrease in cell viability and the presence of necrotic processes shown by Fourier transform infrared analysis, suggesting a direct correlation of prostanoids in cellular homeostasis and survival. models to elucidate the effects of the drug on COX-2 activity and its consequence to the heart. Material and Methods Cell culture and celecoxib treatment H9c2 is a rat embryonic cardiac myoblast cell line (ATCC: CRL-1446) obtained from Banco de Clulas do Rio de Janeiro, Brazil. Cells were routinely grown in Dulbecco’s modified Eagle’s medium (Gibco, USA), containing 2 mM L-glutamine and 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum (Gibco) at 37C in a 5% CO2 atmosphere. Celecoxib (Celebra, C17H14F3N3O2S, 381.373 g/mol; Pfizer Inc., USA) was reconstituted in 1% dimethyl sulfoxide (DMSO). In the control assays (0 M celecoxib), DMSO was also added at the same concentration (1%). Cell viability assay The effect of celecoxib on cell viability was determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This method measures mitochondrial activity based on Tenapanor the reductive cleavage of yellow tetrazolium salt to a purple formazan compound by the dehydrogenase activity of intact mitochondria. The methodology was performed by adapting the protocol described in Mosmann (20). Cells (1.5104) were grown on 96-well plates and exposed to 0, 1, 10, and 100 M celecoxib for 24 h. The culture medium was replaced by a fresh one containing 0.5 mg/mL MTT (Sigma-Aldrich, Germany) and cells were incubated for an additional 2 h. The medium was removed and the formazan product was dissolved in 200 L DMSO for 30 min under gentle agitation. The plates were read with a 570-nm filter in an ELISA Spectracount Reader (Packard Instrument Company Inc., USA). Fluorescence microscopy and DNA analyses Fluorescence microscopy analyses were performed with 3.0104 cells per group, previously grown on sterile coverslips. After treatments (0, 1, 10 M celecoxib), cells were fixed and permeabilized Tenapanor with 3.8% paraformaldehyde in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 7 min. Next, cells were incubated in a PBS solution containing 3.33 ng/L 4,6-diamidino-2-phenylindole (DAPI) at room temperature for 5 min for nuclei staining. Finally, cells were washed three times with PBS for 5 min, mounted on slides using 200 mM propyl gallate (Sigma-Aldrich) in 90% glycerol, and subjected to microscopic analysis. Images were taken with a Leica photomicroscope (DMLB) equipped with an HBO 100-W mercury lamp and the corresponding ultraviolet fluorescence microscopy filter. Tenapanor The statistical analyses were performed by counting 100 cells. RNA extraction and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) H9c2 cells were incubated with celecoxib (0, 1, 10 M) for 24 h and then used for RT-PCR analysis. The total RNA was extracted from 1106 cells according to the instructions from the supplier of the Trizol reagent (Life Technologies?, USA) and then quantified using NanoDrop_1000 (Thermo Scientific, USA). One microgram of each RNA sample was reverse-transcribed into first-strand cDNA using the cloned AMV Reverse Transcriptase (Life Technologies?) following instructions from the supplier. cDNAs were used as templates in RT-PCR analyses, and specific genes were amplified using the following sets of primers (Life Technologies?): -actin, 5-gatcatgtttgagaccttcaacac and 5-cgtcacacttcatgatggagttga; bcl2, 5-tttgagttcggtggggtcat and 5-tgacttcacttgtggcccag; Bax, 5-tggcagctgacatgttttctgac and 5-gtcccaaccaccctggtcttgg; caspase, 5-gcacacattatagctactgg and 5-gttaaactccgacgacgtatta; caspase-8, 5-cgatattgctgaacgtctgg and 5-ctgcaagacaactcgagc; caspase-9, 5-cgtggtggtcattctctctca and 5-cttgacactgcgtccagctg; Cox-2, 5-tccagatcacatttgattgacag and 5-tctttgactgtgggaggataca; c-Fos, 5-gacagatcaacttgaagacg and 5-ggtgaagacaaaggaacg. PCRs were carried out using appropriate annealing temperatures, and the products were analyzed on 1% agarose gels stained with ethidium bromide, and photographed using Kodak Gel Logic 100 Imaging System (USA). The bands were quantified with the Quantity One Software, Bio-Rad (USA). Western blot analyses and PGE2 immunoassays Protein samples.