RNU48 expression was used as an internal control. Beta-actin levels were used as loading control. All oligonucleotide transfection experiments PD332991 were performed in triplicate. For chromatin immunoprecipitation assays, chromatin fragments, derived from untreated, sicontrol- or siPD1-treated Jurkat cells, were immunoprecipitated with 8 g of antibody against STAT5 (ab7969, AbCam). DNA extraction was performed using Qiagen Purification Kit. Real-time PCR analysis was performed for miR-21 (forward: 5′-AGGGGACAAGTCAGAGAGAGG-3′ and reverse: 5′-TCCTCAGAGTAAGGTCA GCTCAG-3′. As a negative control, Jurkat cells were transfected with an siRNA against STAT5, resulting in inhibition
of STAT5 expression levels (90–95% decrease). Using these cells, ChIP was performed followed by PCR analysis. In addition, a positive control was used in the same experiment. Specifically, STAT5 ChIP was performed followed by PCR analysis for CISH (cytokine inducible SH2 protein) gene, a known direct target of STAT5 in human cells 40. The PCR primers used for CISH
were 5′-CTATTGGCC CTCCCCGAC-3′ (forward) and 5′-AGCTGCTGCC TAATCCTTTG-3′ (reverse). As a negative control, a noncontaining STAT5-binding site region was used. The PCR primers used were as follows: forward: 5′-GGTCAGGAGATTGGGA CCAT-3′ and reverse: 5′-TGTGCCTCCTGGGTTCAT-3′. The miRNA database miRBase (http://microrna.sanger.ac.uk/), the PicTar database (http://pictar.bio.nyu.edu/), and the TargetScan version 4.2 (http://www.targetscan.org/index.html) databases were used to identify the potential miRNA targets. In order to have more accurate prediction results, we chose the target genes LBH589 clinical trial that were predicted in two out of the three databases and were Interleukin-2 receptor conserved in other species. Jurkat cells were seeded in 24-well plates and were transfected using Lipofectamine 2000 (Invitrogen). Firefly luciferase reporter gene constructs containing the 3′UTR of PDCD4 (PDCD4-luc) were transfected together with 100 nM microRNA negative control or miR-21. Cell extracts were prepared 24 h after transfection, and the luciferase activity was measured using the Dual
Luciferase Reporter Assay System (Promega, WI, USA). Statistical analysis was performed using either ANOVA or the nonparametric Mann–Whitney U-test. Results were expressed as mean±SEM and p-values <0.05 were considered as statistically significant. The authors thank G. Bertsias for critical review of the manuscript, S. Jaeger for using bioinformatic tools to identify STAT5 sites in microRNA promoters and C. Choulaki for technical assistance. The authors acknowledge the Dana Farber Microarray Facility for performing the microRNA array experiments. This work was supported by the European Union’s Six Framework (FP6) Autocure project and the Hellenic Society of Rheumatology. Conflict of interest: The authors declare no financial or commercial conflict of interest.