mTORC2 suppresses c-Myc/pri-miR-9-2/miR-9-3p in an Akt-independent manner. (A and B) MCF-7 cells were transfected with vector, pUSE-Akt CA, or pUSE-Akt DN, as indicated. 24 h after transfection, cells were treated with or without PP242 for an additional 24 h, harvested, and monitored to determine the miR-9-3p level using RT-qPCR (A) or analyzed for c-Myc expression and PARP cleavage using Western blotting (B). MCF-7 cells were cotransfected with pEGFP-N1 and pUSE-Akt CA (vector) and treated as in A, followed by FACS sorting. (C and D) Sorted cells were analyzed for pri-miR-9-2/miR-9-3p levels (C) or c-Myc expression and PARP cleavage (D). (E–H) MCF-7 (E and F) and A549 (G and H) cells were transfected with a pool of two different siRNAs of Akt1 (1:1). (E and G) 36 h after transfection, cells were serum starved or treated with 400-µM 5-FU for an additional 24 h and analyzed for pri-miR-9-2 (left), miR-9-3p (middle), or c-Myc expression and PARP cleavage (F and H). Error bars represent mean values ± SEM. C, control; NC, negative control; V, vector.