Supplementary Figure Legends
Supplementary Figure 1- PLGF/VEGF induces FLT-1 accumulation in cell
membrane protrusions with minor effects on leukemia cell survival. (A) PLGF and
VEGF stimulation for 24 hours of leukemia cells promoted a minor increase in
leukemia cell viability. These results were repeated three times and the error bars depict
the standard error of the mean. (B) Leukemia cells were attached to poly-L-lysine
coated slides, left untreated or stimulated with PLGF/VEGF for 15 minutes (100 ng/mL
and 50 ng/mL, respectively), fixed and immuno-stained with anti-FLT-1 antibody. An
FLT-1 inhibitor (IR1) was used to demonstrate the specificity of PLGF effects.
Confocal analysis of FLT-1 membrane protrusions in HEL cells are indicated by white
arrows (z-projections of 0.5 m optical sections).
Supplementary Figure 2-- PLGF/VEGF induces co-localization of FLT-1 with
polymerized actin in HEL cells. (A) Co-localization of FLT-1 (red) with polymerized
actin (green) in membrane protrusions was assessed by immunofluorescence after cell
stimulation with PLGF for 30 minutes. (B) After immunofluorescence staining, 6
distinct powerfields were chosen for each condition (control and PLGF stimulated cells)
and the number of cells with protrusion formation of FLT-1/F-actin were counted and
represented in relation to control cells.
Supplementary Figure 3- PLGF stimulation promotes FLT-1:Hsp90 association to
actin-cytoskeleton but not with microtubules. HEL cells were stimulated or not with
PLGF and total cell extracts were prepared and immunoprecipitated with anti--tubulin
antibody and analysed by SDS-Page. Blots were incubated with anti-FLT-1 and antiHsp90 antibodies. The results are representative of 3 independent experiments.
Supplementary Figure 4- Caveolin binding domains in FLT-1 aminoacid sequence.
Four potential caveolin-binding sites were found throughout the entire Human FLT-1
sequence (underlined and in grey) in the total protein amino acid sequence of human
FLT-1 (SWISSPROT database search).
Supplementary Figure 5- PLGF stimulation of AML cells induces caveolin-1
protein expression and interaction with FLT-1. (A)- Total leukaemia cell extracts
were analysed by SDS-Page and after western blolt analyses an increase in caveolin-1
protein expression was detected in AML cells after PLGF stimulation. (B) Coimmunoprecipitation of caveolin-1 from HEL total extracts showed an in vitro
association with the molecular chaperone Hsp90, - actin and FLT-1. Total nonimmunoprecipitated cell extracts (50 g) were used as control. The autoradiographs
shown are representative of similar results from three independent experiments.
Supplementary Figure 6- Actin-cytoskeleton destabilization by cytochalasin D
blocks PLGF-induced leukemia cell migration. (A) Leukemia cell treatment with
cytochalasin D at 50 ng/mL decreased significantly PLGF-induced leukemia cell
migration (total number of cells in 5 HPF of a transwell migration system; p<0.05) and
(B) cell survival (p<0.05). Results are representative of 3 independent experiments.
Error bars depict the standard error of the mean.
Supplementary Figure 7- Treatment with Cytochalasin D blocks cell protrusion
formation and interactions of caveolin-1 with FLT-1, HSP90 and actin
cytoskeleton. (A) Treament of leukemia cells with cytochalasin D for 30 minutes
stopped protrusion formation in PLGF-induced cells (z-projections of 0.5 m of
confocal optical sections; red- FLT-1, green- phalloidin-FITC) and (B) blocks the
interaction between FLT-1, -actin, Hsp90 and caveolin-1 at the cell membrane found
with immunoprecipitation assays with anti-caveolin-1 antibody.
Supplementary Figure 8- Microtubular destabilization with Taxol does not affect
membrane protrusion formation induced by PLGF stimulation. Leukemia cells
were attached to poly-L-lysine coated slides, and were treated with taxol, a microtubulepolimerizing agent (taxol at 100 ng/mL), fixed and immuno-staining with anti-FLT-1
antibody or double-stained with mouse anti--tubulin antibody. Treatment of leukemia
cells with does not stop PLGF-induced formation of membrane protusions z-projections
of 0.5 m of confocal optical sections; red- FLT-1, green- -tubulin).