Figure 7. mTORC2 suppresses c-Myc/miR-9-3p via PP2A-mediated dephosphorylation of c-Myc. (A) MCF-7 cells were transfected with nontargeted siRNA (NC) or two siRNAs targeting different regions of Rictor mRNA. After 60 h, cells were harvested for Western blot analysis. (B) 60 h after transfection as indicated, MCF-7 cells were lysed and immunoprecipitated with PP2A (top) or c-Myc (bottom) and assayed for PP2A activity. For the latter, PP2A activity was normalized to immunoprecipitated c-Myc levels. (C) MCF-7 lysates were immunoprecipitated with antibodies against Rictor or PP2Ac and blotted for Rictor, PP2Ac, and CIP2A. (D) 200-nM PP242–treated MCF-7 cells were lysed, immunoprecipitated, and blotted as indicated. (E) MCF-7 cells were treated with OA gradient, as indicated, in the presence or absence of serum starvation for 24 h. After 24 h, cells were harvested for Western blotting (top left) or RT-qPCR analysis of miR-9-3p level (top right). MCF-7 cells were transfected with siRNA for PP2Ac (siPP2A). After 60 h, cells were harvested for Western blotting (bottom left) or RT-qPCR analysis of the miR-9-3p level (bottom right). (F) MCF-7 cells were cotransfected with siRNAs for both Rictor and CIP2A (left) or transfected with siRNA for Rictor followed by 2.5-µM FTY720 treatment for 24 h (right). 60 h after transfection, cells were harvested either for Western blot analysis (top) or RT-qPCR assay of miR-9-3p (bottom). (G) Effects of ectopic expression of WT c-Myc (myc WT) or S62A mutant (myc S62A) on the miR-9-3p level regulated by simultaneous silencing of Rictor and c-Myc were analyzed using RT-qPCR. Error bars represent mean values ± SEM. C, control; IP, immunoprecipitation; NC, negative control; SF, serum free; WB, Western blot.
Figure 7.

mTORC2 suppresses c-Myc/miR-9-3p via PP2A-mediated dephosphorylation of c-Myc. (A) MCF-7 cells were transfected with nontargeted siRNA (NC) or two siRNAs targeting different regions of Rictor mRNA. After 60 h, cells were harvested for Western blot analysis. (B) 60 h after transfection as indicated, MCF-7 cells were lysed and immunoprecipitated with PP2A (top) or c-Myc (bottom) and assayed for PP2A activity. For the latter, PP2A activity was normalized to immunoprecipitated c-Myc levels. (C) MCF-7 lysates were immunoprecipitated with antibodies against Rictor or PP2Ac and blotted for Rictor, PP2Ac, and CIP2A. (D) 200-nM PP242–treated MCF-7 cells were lysed, immunoprecipitated, and blotted as indicated. (E) MCF-7 cells were treated with OA gradient, as indicated, in the presence or absence of serum starvation for 24 h. After 24 h, cells were harvested for Western blotting (top left) or RT-qPCR analysis of miR-9-3p level (top right). MCF-7 cells were transfected with siRNA for PP2Ac (siPP2A). After 60 h, cells were harvested for Western blotting (bottom left) or RT-qPCR analysis of the miR-9-3p level (bottom right). (F) MCF-7 cells were cotransfected with siRNAs for both Rictor and CIP2A (left) or transfected with siRNA for Rictor followed by 2.5-µM FTY720 treatment for 24 h (right). 60 h after transfection, cells were harvested either for Western blot analysis (top) or RT-qPCR assay of miR-9-3p (bottom). (G) Effects of ectopic expression of WT c-Myc (myc WT) or S62A mutant (myc S62A) on the miR-9-3p level regulated by simultaneous silencing of Rictor and c-Myc were analyzed using RT-qPCR. Error bars represent mean values ± SEM. C, control; IP, immunoprecipitation; NC, negative control; SF, serum free; WB, Western blot.

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