Data Supplement from The Lipid Kinase PI4KIIIβ Is Highly Expressed in Breast Tumors and Activates Akt in Cooperation with Rab11a

Fig. S1. siRNA depletion of PI4KIIIbeta abolishes protein immunostaining. Confocal images of PI4KIIIbeta (green) in BT549 cells treated with negative control siRNA or two distinct siRNA sequences targeted to PI4KIIIbeta respectively. Scale bars indicate 10 microm Fig. S2. PI4KIIIbeta-mediated Akt activation is independent of serum growth conditions. Western blot analysis showing increased Akt phosphorylation at Ser473 and Thr308 for PI4KIIIbeta- overexpressing cells as compared to vector control cells grown overnight in growth media containing no serum, 1% serum or full (10%) serum. Fig. S3. Lipid antibody immunostaining (red) of PI(4)P at the Golgi and at the plasma membrane in BT549 cells transiently transfected with PI4KIIIbeta-GFP. Scale bars indicate 10 microm. Fig. S4. Inhibition of PI4KIIIbeta by Pik93. Confocal images taken during live cell imaging, following a given BT549 cell transiently transfected with FAPP1-PH-GFP and treated with either DMSO (vehicle control) or Pik93 (250 nM) over 60 min show that the PI(4)P reporter construct is redistributed to the cytoplasm in cells treated with Pik93 at the 30 min mark. This same effect is not observed in cells treated with DMSO. Scale bars represent 10 microm. Fig. S5. Ectopic expression of a catalytically inactive (D656A) PI4KIIIbeta leads to Akt activation in MCF10A cells. Western blot analysis showing increased levels of phosphorylation of Akt at Thr308 and at Ser473 in WT-and two independently derived KD-PI4KIIIbeta-expressing MCF10A cell lines as compared to the vector control. Figure is representative of duplicate experiements. Fig S6. (A) Upper panels. Confocal images of Rab11a (green) and PI4KIIIbeta (magenta) in vector control and WT-and KD-PI4KIIIbeta-expressing BT549 cells (from Figure 6). Scale bar represents 1 microm. Lower panels. Intensity profile for Rab11a (green) and PI4KIIIbeta (magenta) across the yellow line drawn in the upper panel. Rab11a vesicles containing PI4KIIIbeta are marked with an asterisk. (B) The percentage of Rab11a vesicles that contain visible PI4KIIIbeta in each of the cell types. At least 50 vesicles per cell were counted in random peripheral fields and the number of cells counted for each type is shown. Significant differences (Students t test, p<5 x10-12) are indicated by a line and double asterisk. (C) The Pearson's correlation coefficient (r) for PI4KIIIbeta and Rab11a in whole BT549 cell lines. Data is presented as the mean {plus minus} SE of the mean for 15 cells imaged from three independent experiments for each cell line. Statistical significance (P < 0.01, Student's t-test) is indicated by (**). Fig S7. (A) Upper panels. Confocal images of Transferrin Receptor (TfR; magenta) and PI4KIIIbeta (green) in vector control and WT-and KD-PI4KIIIbeta-expressing BT549 cells (from Figure 6). Scale bar represents 1 microm. Lower panels. Intensity profile for TfR (magenta) and PI4KIIIbeta (green) across the yellow line drawn in the upper panel. TfR vesicles containing PI4KIIIbeta are marked with an asterisk. (B) The percentage of Rab11a vesicles that contain visible PI4KIIIbeta in each of the cell types. At least 50 vesicles per cell were counted in random peripheral fields and the number of cells counted for each type is shown. Significant differences (Students t test, p<0.003) are indicated by a line and double asterisk. (C) The Pearson's correlation coefficient (r) for PI4KIIIbeta and TfR in whole BT549 cell lines. Data is presented as the mean {plus minus} SE of the mean for 15 cells imaged from three independent experiments for each cell line. Statistical significance (P < 0.01, Student's t-test) is indicated by (**).