Supplementary Figures:
Suppl. Figure 1. CXCR7 is not detectable in HUVECs. Confocal
immunofluorescence microscopy showing the expression of (A) CXCR4 but
not (B) CXCR7 in HUVECs. 4',6-diamidino-2-phenylindole (DAPI) was used
for nuclear counterstaining. Scale bars, 20 µm.
Suppl. Figure 2. SDF-1/ CXCR4 blockade inhibits angiogenesis in
HUVECs. (A) Endothelial proliferation and (B) tube formation assay of cells
treated with control IgG or a neutralizing SDF-1 antibody. (C) Western blots of
whole cell lysates for CXCR4 and for the house keeping protein β-actin,
demonstrating that CXCR4 was downregulated in endothelial cells following
by transduction with the shcxcr4 vector. (D) Endothelial proliferation, (E)
transwell migration and (F) tube formation assay of cells transduced with the
lentiviral control vector pLKO.1 or with the vector pLKO.1-shRNA-cxcr4
(shcxcr4) that was used to knock down CXCR4 expression. In (A-B and D-F),
cells were cultured for 3 days (proliferation assay) or 24 hours (migration and
tube
formation
assay).
*p<0.05
versus
control
(n=3-4
independent
experiments).
Suppl. Figure 3. LDL downregulates chemokine receptor CXCR4 on the
protein, but not the mRNA level. (A) Western blots for CXCR4 and for the
house keeping protein α-tubulin of whole cell lysates of endothelial cells
exposed to different LDL concentrations (25-100 μg/ml) for various durations
(6 hours-48 hours). Note the reduced CXCR4 abundance after 48 hours of
LDL exposure. (B) Real-time PCR for cxcr4 mRNA normalized with gapdh
mRNA of endothelial cells exposed to different LDL concentrations (25-100
μg/ml) for various durations (6 hours-48 hours). Note the absence of an
mRNA response following LDL exposure. *p<0.05 versus control (n=3
independent experiments).
Suppl. Figure 4. Oxidized LDL decreases LDL receptor but not CXCR4
abundance. Western blots for (A) CXCR4 and (B) LDL receptor (LDLR) of
whole cell lysates of endothelial cells exposed to various concentrations of
oxidized LDL (oxLDL) or native LDL (nLDL; 100 µg/ml) for 24 or 48 hours.
Note the decreased CXCR4 abundance that was noticed only after 48 hours
of native LDL exposure (shown in A). Furthermore note that both native LDL
and oxidized LDL (100 µg/ml) reduce LDL receptor abundance (demonstrated
in B). *p<0.05/**p<0.01/***p<0.001 versus control; #p<0.05 versus oxLDL (100
µg/ml) (n=3 independent experiments).
Suppl. Figure 5. LDL receptor blockade does not alter CXCR4
degradation. (A) Western blots of whole cell lysates of endothelial cells
showing time dependent decrease of LDL receptor (LDLR) abundance
following LDL exposure (100 µg/ml). Note that LDL receptor abundance
decreases
starting
at
12
hours
after
LDL
incubation.
(B)
Co-
immunoprecipitation with CXCR4 antibody revealing physical interaction of
CXCR4 and LDL receptor after 4 hours of LDL exposure (100 µg/ml).
Unspecific rabbit IgG was used as control. (C, D) Western blots of whole cell
lysates of endothelial cells demonstrating that the delivery of the LDL receptor
inhibitor receptor-associated protein (RAP; 1 µM) one hour before LDL
exposure (100 µg/ml) does not reverse the CXCR4 degradation after 48 hours
(C) or 72 hours (D). (E) Western blots of whole cell lysates of endothelial cells
exposed to LDL (100 µg/ml) for 12-72 hours showing no change in the
abundance of LDL receptor-related protein (LRP)-1. **p<0.01/***p<0.001
versus control (n=3 independent experiments).
Suppl. Figure 6. Inhibition of 37kDa/67kDa laminin receptor does not
change CXCR4 degradation. (A) Western blots of whole cell lysates of
endothelial cells exposed to LDL (100 µg/ml) for 12-72 hours showing no
change in the abundance of 37kDa/67kDa laminin receptor. (B) Western blots
of whole cell lysates of endothelial cells incubated with neutralizing antibody
against laminin receptor (LR) or mouse IgG (as control) one hour before LDL
exposure (100 µg/ml) for 48 or 72 hours demonstrating that CXCR4
abundance does not change in response to laminin receptor blockade.
*p<0.05/**p<0.01/***p<0.001 versus control (n=3 independent experiments).
Suppl. Figure 7. Liver X receptor blockade does not influence CXCR4
degradation. (A) Western blots of whole cell lysates of endothelial cells
exposed to LDL (100 µg/ml) for various durations showing no change in liver
X receptor (LXR)-α/β abundance, whereas CXCR4 abundance decreases
after 48 and 72 hours. (B) Western blots of lysates of endothelial cells
exposed to the LXR antagonist geranylgeranylpyrophosphate (GGPP; 5 or 10
µM) one hour before LDL exposure (100 µg/ml) for 12-72 hours. Note that
CXCR4 abundance does not change in response to LXR blockade.
*p<0.05/**p<0.01/***p<0.001 versus control;
LDL/GGPP 5 µM;
+p<0.05/+++p<0.001
#p<0.05/##p<0.01
versus –
versus –LDL/GGPP 10 µM (n=3
independent experiments).
Suppl. Figure 8. Combined VEGF and SDF-1 treatment does not alter
CXCR4 and VEGFR2 abundance following LDL exposure. Western blots
for CXCR4, VEGFR2 and the house keeping protein GAPDH of whole cell
lysates of endothelial cells exposed to LDL (100 μg/ml), VEGF (8 ng/ml) and
SDF-1α (100 ng/ml), either alone or in combination with each other. LDL was
administrated 48 hours prior to VEGF or SDF-1 treatment that took place 24
hours before cells were harvested. *p<0.05/**p<0.01 versus control;
++p<0.01
versus SDF-1α; ##p<0.01 versus VEGF (n=3 independent experiments).