Fig. 2
PIK3R1 loss activates STAT3 and AKT signaling. a Reverse phase protein array was performed using lysates of seven serous ovarian cancer cell lines transfected with PIK3R1 siRNA for 72 h. Proteins with ≥20% difference in levels between PIK3R1 siRNA-transfected compared to that of mock-transfected cells in at least three PIK3R1-wild-type cell lines are shown. The corresponding total proteins of the phosphorylated proteins are also presented. b Total lysates of transfected SKOV3, OVCAR8 or OAW28 were analyzed with western blotting. ERK2 was loading control. c SKOV3 cells were transfected with siRNA for 48 h prior to treatment with PIK90 (1 μM) or DMSO for another 24 h. Cell lysates were subjected to western blotting. d Representative immunohistochemical images of ovarian cancer specimens stained with p85α, STAT3 pY705 or AKT pT308 antibodies. Nuclei were counterstained in hematoxylin. Scale bar, 100 μm. e SKOV3 cells were transfected with siRNA for 72 h and harvested for subcellular fractionation followed by western blotting. α-Tubulin and Histone H3 were molecular markers for the cytosolic and nuclear fractions, respectively. f SKOV3 were transfected with siRNA for 24 h and immunofluorescence were performed with STAT3 antibody (Texas red) and DAPI (blue). Scale bar, 20 μm. g SKOV3 cells were transfected with PIK3R1 siRNA 24 h followed by co-transfection of 4xM67 pTATA TK-Luc (containing STAT3-binding site) with Renilla luciferase vector. Dual-luciferase reporter assay was performed after 24 h. h SKOV3 transfected with siRNA for 48 h were treated with Stattic (10 μM) or DMSO for 24 h. Total RNA was collected for real-time PCR. Comparative Ct values calculation was performed for the relative abundance of genes with respect to GAPDH expression. The numbers below the blots represent the mean values from densitometry readings of three independent experiments. Error bars represent SD. ***p < 0.001; ns, no significant difference compared with mock using t-test
