RICTOR knockdown reduces β1 integrin-induced AKT Ser473 phosphorylation but ILK or PAK knockdown has no effect.

<p>(A) HeLa cells were transfected with siRNA directed against RICTOR and then allowed to adhere to plates coated with invasin (β1 integrin-ligand) or Pluronic (non-adhesive control) for 60 min. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and quantifications of AKT pSer473, FOXO1 pThr24, and BAD pSer136 on Pluronic and invasin are given below (mean ± s.e.m.; <i>n</i> = 3; * represents <i>p</i><0.005 and ** represents <i>p</i><0.05). (B) MCF7 cells were transfected and treated in the same manner as in (A). (C) HeLa cells transfected with ILK-directed siRNA or non-target siRNA were allowed to adhere to plates coated with Pluronic, collagen type I or invasin. Cells were lysed and analysed as explained above. A representative western blot is shown and the graph to the right shows quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; <i>n</i> = 3). (D) HeLa cells were transfected simultaneously with PAK1- and PAK2-directed siRNAs or non-target siRNA. Adhesion assays were performed on plates coated with Pluronic, invasin or collagen type I. Cell lysates were subjected to SDS-PAGE (10% gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and the graph below presents quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; <i>n</i> = 3). (E) Adhesion assay with MCF7 cells transfected with PAK1- and PAK2-directed siRNAs or non-target siRNA, performed as described in (A).</p

Effects of RICTOR, ILK or PAK knockdown on LPA-induced AKT Ser473 phosphorylation in HeLa and MCF7 cells.

<p>(A) HeLa cells were transfected with RICTOR-directed siRNA or non-target siRNA and then stimulated with LPA (10 µM) for 20 minutes. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) and LPA-induced AKT Ser473 phosphorylation was determined by western blotting. A representative western blot is shown and the graph below provides quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3). (B) HeLa cells transfected with ILK-directed siRNA and stimulated by LPA were analysed as described above. The graph below shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2). (C) MCF7 cells transfected as indicated were stimulated with LPA (5 µM) for 5 min and analysed as described above. A representative western blot is presented and the graph below shows quantified levels of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005). (D) MCF7 cells were transfected simultaneously with PAK1 and PAK2 siRNA or with non-target siRNA and then stimulated with LPA (5 µM) for 5 min. A representative western blot is shown. The graph below is a quantification of AKT pSer473 levels (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (E) HeLa cells were treated with increasing concentrations of the PAK inhibitor IPA3 or with DMSO and then stimulated with LPA (10 µM) for 20 min. The graph below is the quantification of AKT pSer473 levels after LPA-stimulation in cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in LPA-stimulated cells without the inhibitor (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005).</p

PAK is necessary for PDGF but not for EGF-mediated AKT Ser473 phosphorylation whereas RICTOR knockdown inhibits both pathways.

<p>(A) PAK1 and PAK2 expression was suppressed in MCF7 cells using siRNAs and the cells were stimulated with 20 ng/ml PDGF-BB for 10 min. A representative western blot is shown and AKT pSer473 is quantified below (mean ± s.e.m.; <i>n</i> = 2, ** represents <i>p</i><0.05). (B) MCF7 and HeLa cells were transfected as above and stimulated with 20 ng/ml EGF. A representative western blot is shown and below quantification of AKT pSer473 in MCF7 and HeLa cells (mean ± s.e.m.; <i>n</i> = 3) is presented. (C) HeLa cells were treated with PAK inhibitor IPA3 (30 µM) or DMSO as vehicle control and then stimulated with 20 ng/ml PDGF-BB or EGF. A representative western blot is shown. The graph provides quantification of AKT pSer473 levels, after EGF-stimulation of cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in EGF-stimulated cells without the inhibitor (mean ± s.e.m.; <i>n</i> = 3). (D) RICTOR expression was suppressed in HeLa cells using siRNA and the cells were stimulated with EGF (20 ng/ml). Cell lysates were subjected to SDS-PAGE followed by western blotting using antibodies as indicated. A representative blot is shown and quantification of AKT pS473 levels is found below (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (E) HeLa cells transfected with RICTOR-directed siRNA were stimulated with 20 ng/ml PDGF-BB and analysed as in (D). A representative western blot is shown and the graph below shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2, * represents <i>p</i><0.005). (F) HeLa cells, transfected with non-target or ILK-directed siRNA, were stimulated with 20 ng/ml EGF and the samples analysed as explained above. The graph shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2).</p

Adhesion-induced RICTOR-mediated AKT Ser473 phosphorylation promotes cell-survival.

<p>(A) MCF7 cells transfected with RICTOR-directed or non-target siRNA were grown on glass coverslips in complete culture medium for 48 h, then serum starved for 24 h. A TUNEL assay was performed to visualise apoptotic cells and DAPI was used to stain nuclei. Images shown are representative of pictures taken in three independent experiments. (B) Quantification of (A): Five fields were photographed and at least 400 cells were counted from each coverslip and data is presented as fold change in the percentage of apoptotic cells (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (C) MCF7 cells were transfected as described in (A) and after culturing them for 48 h, the cells were trypsinised and 5×10⁵ cells were seeded in starvation medium on invasin- or collagen type I-coated glass coverslips. The cells were incubated in starvation medium for 24 h, fixed, stained with DAPI and observed under a fluorescent microscope. Cells with pyknotic nuclei (condensed and brightly blue-stained) were considered as apoptotic cells. The result is presented as fold change (mean ± s.e.m.; <i>n</i> = 2, * represents <i>p</i><0.005 and ** represents <i>p</i><0.05). (D) MCF7 cells were transfected as explained above and subsequently 1×10⁵ cells were seeded onto collagen type I-coated dishes in starvation medium. The cells were trypsinised after the indicated time periods and the number of viable cells was counted using the Trypan blue exclusion method. The plot shows fold change in the percentage of viable cells (mean ± s.e.m.; <i>n</i> = 2 and ** represents <i>p</i><0.05) normalised to non-target transfected cells for each time point. The actual numbers of viable cells are given in the table below.</p