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Supplementary figure legends
Fig. S1. siRNA depletion of PI4KIIIβ abolishes protein immunostaining. Confocal images of
PI4KIII (green) in BT549 cells treated with negative control siRNA or two distinct siRNA
sequences targeted to PI4KIIIβ respectively. Scale bars indicate 10 μm
Fig. S2. PI4KIIIβ-mediated Akt activation is independent of serum growth conditions. Western
blot analysis showing increased Akt phosphorylation at Ser473 and Thr308 for PI4KIIIβoverexpressing 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 PI4KIIIβ-GFP. Scale bars indicate 10 μm.
Fig. S4. Inhibition of PI4KIIIβ 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 μm.
Fig. S5. Ectopic expression of a catalytically inactive (D656A) PI4KIII 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-PI4KIIIβ-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 PI4KIII (magenta) in
vector control and WT-and KD-PI4KIIIβ-expressing BT549 cells (from Figure 6). Scale bar
represents 1 μm. Lower panels. Intensity profile for Rab11a (green) and PI4KIII (magenta)
across the yellow line drawn in the upper panel. Rab11a vesicles containing PI4KIII are marked
with an asterisk. (B) The percentage of Rab11a vesicles that contain visible PI4KIII 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 PI4KIII and Rab11a in whole BT549 cell lines. Data is presented as the mean ± 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
PI4KIII (green) in vector control and WT-and KD-PI4KIIIβ-expressing BT549 cells (from
Figure 6). Scale bar represents 1 μm. Lower panels. Intensity profile for TfR (magenta) and
PI4KIII (green) across the yellow line drawn in the upper panel. TfR vesicles containing
PI4KIII are marked with an asterisk. (B) The percentage of Rab11a vesicles that contain visible
PI4KIII 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 PI4KIII and TfR in whole BT549 cell lines. Data is presented as
the mean ± 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 (**).