The depleted RNA was fragmented using

The depleted RNA was fragmented using RNase III in preparation for amplification of the cDNA library, which was produced by reverse transcription from adapters attached to the ends of the RNA molecules, in accordance with the SOLiD™ Total RNA-Seq kit protocol (Life Technologies™, CA). Next, 6% denaturing polyacrylamide gel electrophoresis was performed and fragments of appropriate sizes (150 to 250 bases) were cut from the gel for cDNA amplification using PCR. Following recommended protocols, the cDNA was purified and the sizes were confirmed using 2% agarose electrophoresis. The PCR amplification in emulsion was performed using primers complementary to the adapters, in accordance with the Applied Biosystems SOLiD™ 3 Plus System Templated Bead Preparation Guide. After amplification, the microspheres were deposited onto slides for sequencing in accordance with the manufacturer’s recommendations. The SOLiD™ 3 Plus system was used to sequence the 50-nucleotide RNA reads [3].
Acknowledgements This work was part of the Rede Paraense de Genômica e Proteômica supported by Fundação de Amparo a Pesquisa do Estado do Pará and Computational biology CAPES No. 051/2013. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and CNPq grant #482799/2013-7.
Data A 5′-rapid amplification of cDNA ends (RACE) assay revealed a transcription start site located 212 nucleotides upstream of the DDIAS translation start site or 32 AUY922 pairs (bp) downstream of the site reported in the National Center for Biotechnology Information (NCBI) database (Fig. 1). This transcription start site was confirmed via RNA polymerase binding in a chromatin immunoprecipitation (ChIP) assay (Fig. 2). Putative transcription factor binding sites are located in the P3 region of the DDIAS promoter (Fig. 3A). In a P3-luciferase reporter assay, only NFATc1 knockdown significantly suppressed the promoter activity (Fig. 3B and C). DDIAS mRNA expression was found to be regulated by NFAT signaling, as shown by treatment with cyclosporine A (CsA), a calcineurin inhibitor, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore A23187 (Fig. 4). Treatment with PMA andA23187 increased the cellular expression of DDIAS mRNA (Fig. 4A). In contrast, the cellular DDIAS mRNA level decreased after treatment with CsA (Fig. 4B). Previously, we showed that DDIAS knockdown inhibits the growth of lung cancer cells [2]. In the pancreatic cancer cell line PANC-1, NFATc1 knockdown clearly induces DDIAS depletion and results in growth inhibition, indicating the NFATc1-mediated control of DDIAS expression (Fig. 5).
Experimental design, materials and methods
Acknowledgements This work was supported by National Research Foundation (NRF) Grants (2013R1A2A2A01069026) and the KRIBB (grant number KGS4791511) Initiative program of the Korea Research Council of Fundamental Science and Technology.
Data, experimental design, materials and methods
Value of data
Data
Experimental design, materials, and methods To check for statistical significance, the data were evaluated by the analysis of variance (ANOVA) in combination with variances and standard mean deviations in correlation coefficients. Those data were given in the main article [1].
Acknowledgments The author would like to thank the International Bureau (Contract no. BMBF/DLR - FKZ RUS 10/128) at the PT-DLR and the Federal Ministry of Education and Research of Germany (BMBF) supporting the international cooperation (Grant no. 2010-1.1-203-070-026).
Value of the data
1. Data In this study, we analyzed the native form of ArtinM and its yeast-derived counterpart, in terms of their ability to bind to 255 glycans distributed in a microarray platform, in order to identify whether n-ArtinM and y-ArtinM shared sugar-recognition specificity. Measurement of fluorescence intensity indicated that both preparations bound to N-glycan-related sequences (Fig. 1A and B), with a preference for probes having the core trimannoside Manα1-3(Manα1-6) Man. This binding intensity was enhanced when the probe contained a Fucose residue at the trimannoside core (Table 1 – probe 131); whereas binding was diminished when a similar position in the glycan was occupied by β1-2-linked xylose (probes 130 and 132). Some differences between the two lectin forms were identified in the magnitude of binding to probes 129, 131, 133, 135, 147, 148, 149, 150, 152, 153, 158, 159 and 160. In general, y-ArtinM showed higher fluorescence intensity than n-ArtinM.