FIGURE 1.

Rhes regulates autophagy in PC12 cells. A, RT-PCR analysis of Rhes RNA levels in PC12 cells transfected with serial siRNA transfection protocol. Transfection with Rhes siRNA resulted in a 50% reduction of Rhes RNA compared with scrambled (control) siRNA. B, RT-PCR analysis of GAPDH RNA levels in PC12 cells using a validated GAPDH siRNA, illustrating a similar reduction compared with Rhes, demonstrating that our ability to reduce RNA expression is a function of PC12 transfection efficiency. C, Rhes depletion in PC12 cells results in decreased mTOR activity as well as decreased autophagic flux. Cells were treated with scrambled (control) siRNA or siRNA to deplete endogenous Rhes and placed in medium ± chloroquine (CQ) for 90 min prior to lysis. D, Rhes overexpression in PC12 cells results in mTOR activation and increased autophagic flux. Cells were transfected with Myc- or Myc-Rhes for 48 h and then placed in medium ± chloroquine for 90 min. Quantification is shown at right. mTOR activity is determined by phosphorylation of S6K at Thr-389. Autophagic flux is measured as the amount of LC3-II in the presence of chloroquine minus the amount of LC3-II without chloroquine. *, p < 0.05.