Figure 2

Response of RBMS1 3′UTR and protein expression towards miR-106b. (A) The RBMS1 3′UTR fragment was cloned behind the luciferase reporter gene of the pMIR vector. (B) The reporter gene construct was expressed with the miRNA expression constructs of the miR-17 family or with the empty pSG5 vector as control in the indicated combinations. Results represent the mean of at least four independent experiments performed in duplicates. The dashed line represents the luciferase activity of the empty luciferase reporter plasmid with the empty pSG5 vector which was set to 100% (***p < 0.001). (C) Reporter gene vector containing mutated miR-106b binding site in the RBMS1 3′UTR was co-expressed with miR-106b expression plasmid. Luciferase activity of the reporter vector without miRNA expression was set to 100% (***p < 0.001). (D) DU145 or LNCaP cells were transfected either with control vector or miR-106b expression vector. 48 h post-transfection, the protein expression of RBMS1 was determined by Western blot using ß-actin as loading control. Representative cropped Western Blots of RBMS1 detection after miRNA overexpression in DU145 and LNCaP cells from four independent experiments. Full-length blot is presented in Supplementary Figure S5. (E) For determination of relative RBMS1 downregulation, each four Western Blots of DU145 and LNCaP cells transfected either with control vector or miR-106b expression vector were densitometrically quantified in relation to the corresponding ß-actin band as loading control. The data is shown as mean ± SEM whereas the control lane intensity was set to 1.