mTORC2, but not mTORC1, suppresses miR-9-3p to promote cell survival. (A) MCF-7 cells were transfected with nontargeted siRNA (NC) or two siRNAs targeting different regions of Rictor or Raptor mRNA. After 60 h, cells were harvested and lysed, and the miR-9-3p level was monitored using RT-qPCR (left). Expression of Rictor or Raptor and phosphorylation of S6 or Akt were assessed using Western blotting to ensure siRNA-induced depletion and inhibition of mTORC1 or mTORC2 (right). (B) MCF-7 cells were treated with either pp242 or rapamycin as indicated. At 24 h after treatment, miR-9-3p and its precursor were detected by Northern blotting. (C) MCF-7 cells were subjected to serum (left) or amino acid (AA; right) starvation, and the miR-9-3p level was analyzed along with S6 or Akt phosphorylation. MCF-7 cells transfected with the miR-9-3p antagomir were treated with PP242 or rapamycin, as indicated. (D and E) 48 h after transfection, cells were assayed for apoptosis via trypan blue staining (D) or PARP cleavage using Western blotting (E). Error bars represent mean values ± SEM. C, control; NC, negative control; S, serum.