Our model was constructed around the assumption that an autoregulatory loop involving Hsf1 and Hsp90 plays a central role in the control of thermal adaptation

g glycogen as a carrier. Quantitative PCR of miR-133b and miR-130 was performed with TaqMan microRNA assays from Applied Biosystems using 100 ng total RNA for the reverse transcription “9184477 step. For analysis of the protein-coding gene expression, 2 mg RNA were reverse transcribed using random hexamers and SuperScript III First-Strand Synthesis System for RT-PCR. After reverse transcription all samples were subjected to DNAseI treatment in order to remove contaminating genomic DNA. All samples were analyzed with a 7900HT fast real-time PCR system and subjected to comparative DDCt method by using human acidic ribosomal protein as the internal standard. For further information about all primers see 39- untranslated region cloning, mutation and luciferase reporter assay RNA isolation and quantitative PCR from primary prostate cancer tissue miR-133b, a Potent Proapoptotic Molecule rinsed once with PBS and lysed with passive lysis buffer. Luciferase activity was measured in a Victor Luminometer using the Dual-Luciferase Reporter Assay System from Promega. Total Renilla luciferase activity was calculated by normalizing to firefly luciferase in order to correct for differences in transfection efficiency. Statistics Unless otherwise specified, data shown are representative of at least three independent experiments. Statistical significance was calculated by two-tailed Student’s t-test and p,0.05 was considered significant. Statistical analyses of RT-qPCR data from primary tissue was performed using PASW statistics 18.0.0, GraphPad version 5.00 and Medcalc version 11.0. Kolmogorov-Smirnov, Wilcoxon signed rank test and Spearman correlation were used. All tests were performed two-tailed and p,0.05 was considered significant. Receiver operating characteristic curves were calculated and univariate logistic 518303-20-3 chemical information regression was performed to determine the discriminative power. For survival analyses KaplanMeier approach and Cox proportional hazard regression were used. Supporting Information cancer tissue and normal adjacent tissue. ROC analysis was performed on miR-133b expression. Dotted line indicates an AUC of 0.5. Detailed description of the pSILAC protocol. Acknowledgments We thank Mary Louise Grossman for editing and critical reading of the manuscript. Author Contributions Conceived and designed the experiments: JPP AF BT KJ SHEK JS. Performed the experiments: JPP AF MB MA CP JS. Analyzed the data: JPP AF CP BT HJM JS. Contributed reagents/materials/analysis tools: UJ HM CS HJM. Wrote the paper: JPP AF BT KJ SHEK JS. References 1. Kerr JF, Wyllie AH, Currie AR Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239257. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16: 311. Aggarwal BB Signalling pathways of the TNF superfamily: a doubleedged sword. Nat Rev Immunol 3: 745756. Fischer U, Janicke RU, Schulze-Osthoff K Many cuts ” to ruin: a comprehensive update of caspase substrates. Cell Death Differ 10: 76100. Bartel DP MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281297. Pillai RS, Bhattacharyya SN, Filipowicz W Repression of protein synthesis by miRNAs: how many mechanisms Trends Cell Biol 17: 118126. Garofalo M, Condorelli GL, Croce CM, Condorelli G MicroRNAs as regulators of death receptors signaling. Cell Death Differ. 17: 200208. McCarthy JJ

评论已关闭。