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Supplementary Figure 1 siRNA-mediated depletion of PIKfyve and Vac14.
(a) PIKfyve and Vac14 expression was quantified in A431 cells that had been treated
with scrambled control siRNAs, or siRNAs targeted against PIKfyve or Vac14 for 96hrs
using TaqMan Gene expression assays as per manufacturer’s instructions. GAPDH was
used as an internal control to calculate the CT for each sample. (b) Western
Immunoblotting was conducted on whole cell lysates prepared from A431 samples
treated with siRNAs as described above.
Supplementary Figure 2 PIKfyveYM201636 does not induce apoptosis.
Tunel assays were conducted on A431 cells that had been infected with RFP-SL1344 and
treated with YM201636 for 24hrs and 26hrs respectively as described in Figure 1.
Supplementary Figure 3 PIKfyve(K1831)M does not inhibit homotypic fusion of
macropinosomes.
Newly formed macropinosomes, labelled by culturing cells in the presence of 100µg/ml
dextran-TR (MW10,000) for 4mins, and late endosomes/lysosomes were imaged in 4D as
described in the Materials and Methods. Presented is a montage of selected frames of
macropinosomes in a cell transfected with pPIKfyve(K1831M)-GFP. Total duration and
interval were as indicated.
Supplementary Figure 4 PIKfyve(K1831)M does not inhibit Rab7 recruitment.
FlpIn HEK293 cells were transiently cotransfected with pPIKfyve(K1831M)-GFP and
pmRFP-Rab7 and imaged in 4D using confocal microscopy. Presented is a montage of
selected frames. Total duration and interval were as indicated.
Supplementary Movies
Supplementary Movie 1: Macropinosomes in cells expressing PIKfyve-GFP fuse with
late endosomes and lysosomes. Details in Figure Legend 4 (b).
Supplementary Movie 2: Macropinosomes in cells expressing PIKfyve(K1831M)-GFP
do not fuse with late endosomes and lysosomes. Details in Figure Legend 4 (b).
Supplementary Movie 3: mRFP-Rab7 is recruited to macropinosomes in cells expressing
Macropinosomes is cells expressing PIKfyve(K1831M)-GFP. Details in Supplementary
Figure Legend 4.