washed with PBS, and incubated with anti-AIF antibody (1:50, Santa Cruz Biotechnology) for 1 h 30 min at room temperature. Then, cells were washed with PBS and incubated with the secondary antibody anti-mouse-TRITC conjugated (1:200, Ban- galore Genei) for 45 min at room temperature. Finally, cells were washed with PBS and stained with DAPI (0.5 ug/ml) for 10 min. Fluorescent images were acquired through a Confocal microscope (Carl Zeiss, Germany) [33]. Results Expression of miR-23a, miR-27a and miR-24-2 in HEK293T cells after p(23a,27a,24-2) cluster over expression We first did northern blot analysis to determine the expression of miR-23a, miR-27a and miR-24-2 in HEK293T cells. As shown in Fig. 1A, northern blot analysis for microRNAs showed that only microRNA 27a was abundantly expressed in the mature form in HEK293T cells after over expression of p(23a,27a,24-2) cluster. MicroRNA 23a levels were not detectable and there was no change in the mature form of microRNA 24-2 before and after p(23a,27a,24-2) cluster over expression. miRNA Cluster Induce Apoptosis To better address the expression levels of miR-23a, miR-27a and miR-24-2 in HEK293T cells before and after p(23a,27a,24-2) cluster over expression, we did time course study using semiquantitative PCR. Fig. 1B shows that expression of microRNA 23a was very low whereas the expression levels of microRNA 24-2 and microRNA 27a were significantly high after p(23a,27a,24-2) cluster over expression in HEK293T cells as compared to untransfected HEK293T cells. To further confirm the semiquantitative PCR result, we did SYBR green based real time PCR assay for the precursor form and TaqMan based real time PCR assay for the mature form of miR-24-2 and miR-27a. As inferred from the Fig. 1C (i, ii), there was a very significant increase in the precursor form of both miR-24-2 and miR-27a and there was 1.66 fold increase in the levels of the mature form of microRNA 24-2 and 1.42 fold increase in the levels of the mature form of microRNA 27a (Fig. 1C iii, iv) after p(23a,27a,24-2) cluster over expression in HEK293T cells as compared to untransfected HEK293T cells. p(23a,27a,24-2) induces apoptosis in HEK293T cells A number of studies have demonstrated that microRNAs can act as oncogenes or tumor suppressors [34–35]. Infact, the same Figure 1. Overexpression of p(23a,27a,24-2) in HEK293T cells causes a change in precursor and mature form of mir-23a, miR-27a and miR-24-2. A. Northern Blot depicting change in miRNA expression after p(23a,27a,24-2) transfection (i) U6 loading control (ii) miR-23a (iii) miR-27a (iv) miR-24-2. B. Semiquantitative PCR confirms the increase in the miRNA transcript levels of miR-24-2 and miR-27a but not much change in miR-23a after p(23a,27a,24-2) transfection at 8-, 16- and 24- hours post-transfection, M refers to DNA ladder. The constitutive expression of b-actin is shown as the loading control. C. Real Time PCR for (i) pre-miR-24-2, (ii) pre-miR-27a and TaqMan assay for (iii) Mature miR-24-2, (iv) Mature miR-27a. doi:10.1371/journal.pone.0005848.g001 PLoS ONE | www.plosone.org 4 June 2009 | Volume 4 | Issue 6 | e5848