Figure 5.

Overexpression of miR-7 or miR-153 attenuated the effects of rapamycin in cortical neurons. (A,B) Six to seven days old primary cortical neurons were transduced with adenoviral particles expressing scramble miR, miR-7, or miR-153 and left in culture for additional 48 h. One hour before harvest, cultures were supplemented with 20 nM rapamycin. Equal amounts of total protein from lysates of cortical neurons were analyzed on 10% SDS-PAGE and immunoblotted with antibodies specific for phosphorylated forms of p70S6K, S6RP, and eEF2K. To ensure equal loading, membranes were re-probed against GAPDH. Quantification of the results was performed by scanning densitometry. Note that overexpression of miR-7, as well as that of miR-153 attenuated the effect of rapamycin on the phosphorylation of all proteins examined. (C) Six to seven days old primary cortical neurons were transduced with the same adenoviral particles and after 24 h were exposed to 20 nM of rapamycin for an additional of 24 h. Neuronal viability following 24-h treatment with rapamycin was monitored by the MTT assay. Note that overexpression of miR-7 and/or miR-153 attenuated rapamycin-induced cell death. Bars in all the presented graphs depict mean ± s.e.m. ^**P ≤ 0.01, ^***P ≤ 0.001.