Effects of miR-34a replacement on survival pathways and apoptosis occurrence.

<p>A) SKMM-1 cells were transfected with miR-34a (34a) or scramble miR-NC (NC) and after different times from the transfection were collected for Western blot analysis. Thereafter, the expression and phosphorylation of Erk, the activity and expression of Akt and pro-caspase-6 and -3 expression were evaluated after blotting with specific antibodies, as described in “Material and Methods”. The house-keeping protein α-tubulin was used as loading control. Each point is representative of 3 different evaluations performed in 3 different experiments. B) Scan of the bands associated with pErk-2 expression and Akt activity normalized for total Erk-2 or Akt expression, respectively, and of pro-caspase-3 and pro-caspase-6 expression, normalized with the housekeeping protein α-tubulin in SKMM-1 cells, was performed with ImageJ software. The intensities of the bands were expressed as % of changes based upon determination of arbitrary units (%, mean of three different experiments). Each point is the mean of 3 different evaluations performed in at least 3 different experiments. Bars, s.e.’s. C) SKMM-1 cells after transfection with miR-34a (34a) or scramble miR-NC (NC). The cells were collected after the indicated times from the transfection and apoptosis was evaluated with TUNEL assay by FACScan as described in “Materials and Methods”. Results are shown as percentage of apoptotic cells. Data are the average ‘SD of 3 independent experiments.