Table 1.
Primer sequences employed for real-time PCR.
Fig 1.
The effect of mTOR-siRNA on the levels of mTOR mRNA and protein.
HLE B3 cells were treated with mTOR-siRNA, non-silencing siRNA or control (transfection reagent only), and harvested after 24 h, 48 h and 72 h of transfection. (A) MTOR mRNA was quantified by real-time PCR. The values of mTOR were normalized to Actin and then normalized to control relative value. (B) Representative agarose gel images of the real-time PCR products. (C) MTOR protein levels were examined by Western blot. (Data = Mean ± SEM, *p<0.05, **p<0.01, compared with the control groups).
Fig 2.
The effect of mTOR-siRNA on HLE B3 proliferation.
CCK8 assay was used to determine HLE B3 proliferation. MTOR-siRNA significantly reduced cell proliferation at 48 hours of transfection and this effect was enhanced at 72 hours of transfection. (Data = Mean ± SEM, *p<0.05, compared with the control groups).
Fig 3.
The HLE B3 cells growth curve in the presence of mTOR-siRNA and rapamycin.
mTOR-siRNA inhibited cell growth at 48 hours of transfection and this effect was dramatically enlarged at 72 hours of transfection. Rapamycin significantly reduced cell growth at 72 hours of transfection. (Data = Mean ± SEM, *p < 0.05).
Fig 4.
The inhibition of cell migration by mTOR-siRNA.
(A) Representative images were taken from Scratch assay. (B) TGF-β-induced cell migration was blocked by mTOR-siRNA. The Gap closure was reduced by mTOR-siRNA. (C) Cell migration was assessed using the Byoden chamber in the absence of TGF-β. The mTOR-siRNA transfected HLE B3 cells were seeded into the Boyden chamber and incubated for 48 hours. The cell migration was significantly reduced by mTOR-siRNA at 48 hours. (Data = Mean ± SEM, *p < 0.05).
Fig 5.
MTOR-siRNA reduced the formation of Raptor/mTOR complex and inhibited the phosphorylation of p70S6K.
(A) mTOR-siRNA significantly down-regulated p70S6K mRNA expression at 48 hours of transfection, and this effect was enlarged at 72 hours of transfection. The values of p70S6K mRNA were normalized to Actin mRNA and then normalized to control relative value. (B) The phosphorylation of p70S6K was reduced by mTOR-siRNA after 72 hours of transfection. (C) The interaction of Raptor and mTOR were assessed using co-immunoprecipitation assay. In the anti-mTOR antibody precipitates, the levels of Raptor were drastically reduced by mTOR-siRNA. (Data = Mean ± SEM, **p<0.01, compared with the non-silencing siRNA).
Fig 6.
MTOR-siRNA eliminated the protein interaction of Rictor and mTOR protein and inhibited the phosphorylation of AKT.
(A) HLE B3 cells were treated with mTOR-siRNA, non-silencing siRNA or control and harvested after 24, 48 and 72 hours of transfection. The levels of mRNA were determined by quantitative real-time PCR. The values of AKT mRNA were normalized to Actin mRNA and then normalized to the control value. (B) The phosphorylation of AKT was reduced by mTOR-siRNA after 72 hours of transfection. (C) The interaction of Rictor and mTOR proteins was assessed by co-immunoprecipitation using an anti-mTOR antibody. The precipitates were examined by western blot with anti-Rictor antibody. In the mTOR antibody precipitates, Rictor was undetectable in the samples treated with mTOR-siRNA. (Data = Mean ± SEM, **p<0.01, compared with the non-silencing siRNA).
Fig 7.
MTOR-siRNA blocked EMT induced by TGF-β.
HLE B3 cells were transfected with mTOR-siRNA and 24 hours later cells were treated with TGF-β for 48 hours. Cells were then lysed and subjected to Western blot.