mTORC2 mediates pri-miR-9-2/miR-9-3p/E2F1 signaling and apoptosis via c-Myc. (A) The effect of silencing c-Myc on the pri-miR-9-2/miR-9-3p level was assayed using RT-qPCR (left and middle). Protein expression of c-Myc was analyzed via Western blotting (right). (B) MCF-7 cells were treated with PP242 or rapamycin (left) or transfected with two different siRNAs for Rictor and Raptor (right) as indicated, and their effects on c-Myc expression were monitored using Western blotting. (C) MCF-7 cells were treated with 200-nM PP242 or 10-nM rapamycin for 24 h and subjected to chromatin extraction. Sheared chromatin was immunoprecipitated with a c-Myc antibody and the two binding promoter regions of hsa-miR-9-2 (one of three miR-9-3p–coding DNAs) augmented using RT-qPCR. (D and E) The effect of c-Myc silencing on the pri-miR-9-2/miR-9-3p level regulated by PP242 (D) or Rictor knockdown (E) was analyzed using RT-qPCR. (F–I) The effect of c-Myc silencing on apoptosis induced upon Rictor depletion or PP242 treatment was detected with a light microscope (F), trypan blue staining (G), or Western blotting (H and I), as indicated. Bar, 50 µm. (J) In MDA-MB-231 cells, the effect of c-Myc silencing on apoptosis induced upon 200-nM PP242 treatment (left) or Rictor depletion (right) was detected with Western blot analysis of PARP cleavage. C, control; NC, negative control; TSS, transcription start site. Error bars represent mean values ± SEM.