The impact of TEL-AML1 silencing upon miRNA-494 and miRNA-320a is independent of TEL expression. (A) HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) with TEL or TEL-AML1 plasmids cloned into pcDNA 3.1 according to manufacturer's protocol exactly as explained in Diakos et al.8 Cells were lysed in radio-immunoprecipitation assay buffer containing proteinase inhibitors (Complete, Roche) with subsequent western analysis. Transient expression of proteins was confirmed by using TEL antibody (AB23465-100 Abcam) antibody and the LI-COR detection system (LI-COR Odyssey). (B) An ETV6 (TEL) expressing vector was introduced into REH cells by lipofection (lane 1) and the TEL-AML1 expression was silenced using siRNA (lane 2). The expression of TEL and TEL-AML1 was analyzed by Western blot analysis using an antibody against TEL. An anti-tubulin antibody was used for loading control on the same gel (using the LI-COR system). (C-D) The expression of miRNA-320 (C) and miRNA-494 (D) were analyzed by miRNA Taqman PCR. The black bars represent the results from lanes 1 to 3 for the HEK293T experiment shown in Figure 5A, and the gray bars represent the result from lanes 1 and 2 for the REH cell experiment in Figure 5B. miRNA levels are displayed relative to the empty vector HEK293a result.
