Figure 5
Overexpression of miR-9 in CLL cells. I83-E95 cells, completely methylated for miR-9-3, were transfected with miR-9 mimic or scrambled control oligo. Cells were harvested for functional assays 48 hours after transfection. A, Stem-loop RT-qPCR analysis of miR-9 expression at 48 hours after transfection. ΔCt, Ct miR-9-Ct RNU48. RNU48 was chosen as reference using the 2-ΔΔCT method. B, Cell proliferation of CLL cells in response to overexpression of miR-9 was measured by MTT assay. Column, mean percentage of cell proliferation from three experiments conducted in triplicate; C, Cellular death was measured by Trypan blue exclusion assay. Column, mean percentage of cell death from three experiments conducted in triplicate; D, Cell apoptosis of CLL cells after miR-9 transfection was assessed by flow cytometry using FITC Annexin V and PI staining (left panel: representative result; right panel: values represented mean percentage of cell apoptosis from three experiments performed in triplicate. E, Western blot analysis of NFκB1 (P105/P50) after miR-9 transfection. Total protein was extracted 48 hours after transfection and membranes were probed with antibodies to P105, P50 and Anti-actin. Anti-actin protein was regarded as the endogenous normalizer and the relative NFκB1 (P105/P50) protein level was shown. Error bars represent standard deviation.