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  • prostaglandin receptor br Acknowledgments The authors wish

    2018-11-03


    Acknowledgments The authors wish to acknowledge co-funding of this work by Spain׳s Ministry prostaglandin receptor of Economy and Competitiveness (MINECO-INIA-CCAA) and the European Fund of Regional Development (FEDER, Grant RTA2011-00020-C02-02). The staff at the Central Service for Research Support (SCAI) of the University of Cordoba is also gratefully acknowledged for help with the analysis of the proteins.
    Data Two key signals which regulate the induction of apoptosis are DNA damage and calcium (Ca2+) [1,2]. Despite the common use of cisplatin (CisPL) and Ca2+ ionophores such as A23187 to induce apoptosis in cell culture experiments, limited evidence exists in C2C12 cells. Here, we present data describing the cell death response in sub-confluent C2C12 cells exposed to CisPL or A23187 (Fig. 1).
    Experimental design, materials and methods
    Acknowledgments This research was supported by funds (341256) provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) to Joe Quadrilatero. Darin Bloemberg is the recipient of a NSERC postgraduate scholarship. NSERC did not participate in study design, the collection, analysis and interpretation of data, the writing of the report, or the decision to submit the article for publication.
    Data Table 1 provides the list of primers used to determine the relative abundance of each CLICs in the cardiac tissue [1]. Specificity of the CLIC prostaglandin receptor was evaluated using the cardiac tissue lysates from the clic1, clic4 and clic5 mice. Absence of CLIC specific band in the knock out cardiac lysates indicated the specificity of the antibodies used in this study (Fig. 1). Localization of CLIC1, CLIC4 and CLIC5 was deciphered in endoplasmic reticulum (ER) of neonatal cardiomyocytes using the highly specific antibodies (Fig. 1). CLIC1 (37±1.0%, n=3) and CLIC4 (35±1.6%, n=3) showed higher degree of colocalization to the endoplasmic reticulum whereas CLIC5 (15±1.1%, n=3) showed negligible colocalization (Fig. 2). DmCLIC, an ortholog of CLIC in Drosophila melanogaster, localizes to the mitochondria of cardiac tubes [1] as well. Further, specificity of the DmCLIC antibodies was tested in cardiac tubes of CLIC mutant flies, (Fig. 3).
    Experimental design, materials and methods
    Real time PCR analysis and quantification of various CLIC transcripts Hearts from the 2 month old Sprague-Dawley rat were used to prepare total RNA using TRIZOL reagent (Life technologies). This was followed by double digestion with RNase-free DNase for 30min at 37°C. RNA prepared was further cleaned up with RNeasy mini kit (Qiagen). Purified RNA (2μg) was reverse-transcribed with Omniscript Reverse Transcription (RT) kit (Qiagen) using oligodT primer. The reverse transcriptase was inactivated by heating at 95°C for 5min. Real-time qPCR was performed using iQ SYBR Green Supermix (Bio-Rad) in applied biosciences system (iQ cycler, Applied Biosystems), according to MIQE guidelines [5,6]. 1μl of RT reaction product, and 0. 3µl of 25pmole/µl primer pairs (Table 1) were added in a 20-μL reaction. Primer pairs were designed to flank an intron to control contamination from genomic DNA. The amplification conditions comprised an initial denaturation step at 95°C for 5min, and 40 cycles of 95°C for 45s, 60°C for 45s, and 72°C for 45s. Mock cDNA (no reverse transcriptase) as well as primers to amplify GAPDH were used as a control to assess the quality of the cDNA and genomic DNA contamination. GAPDH will be used to normalize the expression of different CLICs. All samples were run in triplicates. Threshold cycle values (Cq) were measured at a fluorescence of 100a.u. Efficiency of the primers was calculated using the slope of the standard curve plot (threshold cycle vs. log of various DNA concentration) defined as (10–1/slope–1)×100. Clear single peaks at their melting temperature and a clear band at the expected size in agarose gels confirmed the amplification of specific products.