miRNA Cluster Induce Apoptosis 2 (TRAF 2) and the Fas-associated death domain protein (FADD) are important effectors of TNFR1 signaling, we examined whether miR-23a,27a,24-2 over expression induces apoptosis through TNFR1. Results in the Fig. 5C show that apart from reducing FADD protein, over expression of miR-23a,27a,24-2 cluster in HEK293T cells was causing significant increase in TRAF2 protein levels both in presence or absence of TNF-a. Our western blot data also showed non significant changes in RIP protein levels both in presence or absence of TNF-a. In literature, it has been found that in RIP and TRAF-2 deficient cells, RIP is most likely responsible for NF-kB activation, whereas TRAF-2 preferentially activates c-Jun N-terminal kinase (JNK) [43]. Since, in our study we observed increased TRAF2 and no change in RIP expression, we postulate that miR-23a,27a,24-2 cluster might be inducing apoptosis in HEK293T cells by activating JNK and inhibiting NF-kB. To prove this, we did western blot analysis for p-JNK and NF-kB. (Fig. 6A, C) and also checked for NF- kB transcriptional activity using a reporter assay with pNF-kB-luc plasmid (Fig. 6B). Interestingly, we observed activation of JNK and inhibition of NF-kB in miR-23a,27a,24-2 transfected HEK293T cells as compared to untransfected HEK293T cells. It seems that miR-23a,27a,24-2 induces apoptosis in HEK293T cells via JNK activation. Discussion The recent report by Gu et al [37] showed that upregulation of miR-23a,27a,24-2 cluster decreases transforming growth Figure 5. Sensitivity to apoptosis is increased after transfection of p(23a,27a,24-2). A. Annexin V-PE binding in HEK293T cells i) untransfected cells, ii) cells transfected with 4 mg p(23a,27a,24-2) for 24 h, and iii) cells treated with 20 ng TNF-a iv) cells transfected with 4 mg p(23a,27a,24-2) for 24 h and treated with 20 ng TNF-a. B. Mitochondrial membrane potential in HEK293T cells detected by DiOC6 staining and flow-cytometry analysis, i) untransfected cells, ii) 24 h after transfection with 4 mg p(23a,27a,24-2), iii) treated with 20 ng TNF-a, and iv) 24 h after transfection with 4 mg p(23a,27a,24-2) and treated with 20 ng TNF-a. C. Effect of overexpressed p(23a,27a,24-2) on the protein expression of FADD, pro-caspase 8, TRAF2, RIP. GAPDH/b-actin was used as an internal control. Data are representative of a typical experiment repeated three times with similar results. doi:10.1371/journal.pone.0005848.g005 PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5848