Figure 6. PI3KC2b protects NIH3T3 cells against anoikis. (A) NIH3T3-V, -C2b-DN and -C2b-WT cells were plated in presence of serum on ultra-low attachment matrix. After 16 h Caspase 3/7 activity was measured as readout for detachment-induced apoptosis (anoikis). Data are mean 6 SD of two independent experiments. WT1, 22 and DN1, 22 indicate individual clones. (B) NIH3T3 cells transiently transfected Role of PI3KC2b in RhoA Activation with the wild-type Myc-PI3KC2b, constitutively active form of GFP-fused RhoA protein (CA-RhoA-GFP) or myristylated (Akt-myr) were plated 48 h post-transfection on ultra-low attachment matrix. 16 h later Caspase 3/ 7 activation was analysed as readout for anoikis. Data are mean 6 SD of three independent experiments. Expression level of the transfected proteins and associated signalling was analysed 48 h post-transfection by immunoblotting with indicated antibodies. (C) NIH3T3-C2b-WT cells were transiently transfected with dominant-negative RhoA-GFP (DN- Rho-GFP) or control vector (V). 48 h post-transfection cells were plated on ultra-low attachment matrix and caspase 3/7 activation was measured. Data are mean 6 SD of three independent experiments. 48 h post-transfection expression level of the transfected proteins was analysed by western blot with indicated antibodies. doi:10.1371/journal.pone.0044945.g006 of GFP-fused RhoA and myristylated Akt were used. Cells were grown for 48 h in attachment then trypsinized and washed once with DMEM/10% FCS. The cells were then placed in Ultralow attachment 96-well plates (Corning) at 26105 cells/well in 100 ml DMEM containing 10% FCS at 37u/5% CO2. After 16 h detachment-induced apoptosis was analysed based on the caspase 3/7 activation using the Caspase-Glo 3/7 Assay (Promega), according to manufacturer’s instruction. Immunofluorescence Cells were seeded on glass coverslips and grown for 48 h. Coverslips were rinsed in PBS and fixed with 4% paraformalde- hyde. Prior to permeabilisation with 0.2% Triton X-100, cells were incubated with 0.1M glycine. After blocking in 2% horse- serum immunostaining was performed with an anti-PI3KC2b antibody followed by FITC-conjugated goat anti-rabbit antibody and Alexa Fluor 555 conjugated phalloidin (Invitrogen). Cover- slips were mounted in Mowiol (Centre for Microscopy and Image Analysis, Zurich) and cells were examined by confocal laser scanning microscopy (Leica SP1, Centre for Microscopy and Image Analysis, Zurich). Luciferase Reporter Assays Transient transfections of NIH3T3 cells were carried out using Lipofectamine (Invitrogen) according to the manufacturer’s protocol. Around 56104 cells per well in 12-well plate format were transfected with 25 ng of p3DA-Luc and 50 ng of pRL-TK (Promega, Madison, WI). After transfection cells were maintained in 0.5% FCS for 24 h before stimulation with 15% serum for seven hours. Firefly luciferase activity was normalized to pRL-TK luciferase activity. Figures show mean 6 SD of three independent experiments. Statistical Analysis Analysis of variance and Bonferroni multiple comparison test or Kruskal-Wallis non-parametric analysis of variance test were used to assess statistical significance of differences between groups. P values ,0.05 were considered as significant. Results PI3KC2b Interacts with Dbl The small GTPases Rac and RhoA are regulated by proteins modulating their GTPase activity. We and others reported the interaction of PI3KC2b with RhoGEFs such as Eps8 and intersectin [17,40]. To elucidate the molecular mechanism of PI3KC2b–dependent Rac and RhoA activation in NIH3T3 cells, we investigated the binding of PI3KC2b to a panel of RhoGEFs family members. We performed co-immunoprecipitation studies using an anti-PI3KC2b and an anti-epitope-Glu-tag (EE) antibody PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44945