Supplemental Information
CD44 functions in Wnt signaling by regulating LRP6 localization and
activation
Mark Schmitt, Marit Metzger, Dietmar Gradl, Gary Davidson and Véronique Orian-Rousseau
Figure S1: CD44 is required for activation of β-catenin in HCT cells and for cytoplasmic
accumulation of β-catenin in HeLa cells.
Figure S2: CD44 amplifies Wnt/β-catenin signaling independently of HA and HA binding to
CD44 .
Figure S3: rCD44v4-v7∆Cyt is expressed at the membrane and mutation of the ERM binding
domain reduces CD44 mediated enhancement of Wnt signaling.
Figure S4: CD44 augmentation of Wnt/β-catenin signaling is independent of RTK signaling
via the MEK/Erk- and PI3K/Akt-pathways.
Figure S5: The complex between CD44 and LRP6 occurs through the cytoplasmic domains
and is dependent on the binding of CD44 to ERM proteins.
Figure S6: xCD44-MO injected embryos show a musculature defect phenotype that can be
rescued by hCD44s.
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Fig. S1 CD44 is required for activation of β-catenin in HCT cells and for cytoplasmic
accumulation of β-catenin in HeLa cells.
A) HCT116 cells were transfected with control siRNA or siRNA against all CD44 isoforms
and treated either with Co-CM or Wnt3a-CM for 3 hrs. Cells were subjected to WB analysis
72 hrs after siRNA transfection. Numbers indicate the fold intensity values of the active βcatenin bands normalized to the bands of total β-catenin. B) HeLa cells were transfected with
control siRNA or siRNA against all CD44 isoforms for 72 hrs and treated either with Co-CM
or Wnt3a-CM for 6 hrs. Afterwards, cells were lysed and subjected to subcellular
fractionation. Cell fractions were analyzed by WB analysis. The relative purity of the
membrane and cytosolic fractions was confirmed by sequential probing for the cytoplasmic
protein Erk and the membrane protein CD44. Subcellular fractionation and WB analysis are
described in “Materials and Methods”.
Fig. S2 CD44 amplifies Wnt/β-catenin signaling independently of HA and HA binding to
CD44 .
A) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together
with an empty vector or a cDNA for hCD44s (75 ng). Following stimulation with Wnt3a-CM or
Co-CM (20 hrs) in the presence or the absence of hyaluronidase (HAse, 50 U/ml) cells were
lysed and subjected to luciferase measurements. In parallel, untreated or HAse treated
HEK293 cells were stained with a biotinylated HA binding protein and Alexa488 coupled
streptavidin. Representative pictures were taken using a Leica SP5 fluorescence microscope
with a 63X objective (scale bar = 25 µm). B) HEK293 cells were transfected as in A). After
stimulation with Wnt3a-CM or Co-CM (20 hrs) in the presence or absence of antibodies
blocking the interaction between HA and CD44 (BU52) or IgG (3 µg/ml), cells were lysed and
subjected to luciferase measurements. C) HEK293 cells were transfected with TOPFlash and
control TK-Renilla vectors together with empty vector or 75 ng of cDNAs for hCD44s or
hCD44s mutated in the HA binding domain (hCD44s-HAmut). After stimulation with Wnt3a2
CM or Co-CM (20 hrs) cells were lysed and subjected to luciferase measurements or in
parallel to WB analysis. Reporter gene assays and WB analysis are described in “Materials
and Methods”. All data represent mean +/- SD from at least 4 independent experiments
performed in triplicates. Statistical significance was analyzed using the student´s t-test (*p<
0.05).
Fig. S3 rCD44v4-v7∆Cyt is expressed at the membrane and mutation of the ERM
binding domain reduces CD44 mediated enhancement of Wnt signaling.
A) HEK293 cells were transfected with an empty vector or cDNA for rCD44v4-v7∆Cyt. 48 hrs
later, cells were lysed and subjected to subcellular fractionation. Cell fractions were analyzed
by WB analysis. The relative purity of the membrane and cytosolic fractions was confirmed
by sequential probing for the cytoplasmic protein Erk and the membrane protein transferrin
receptor. Subcellular fractionation and WB analysis are described in “Materials and
Methods”. B) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors
together with an empty vector or cDNAs for CD44s (75 ng) or CD44s mutated in the ERM
binding domain (CD44s-ERMmut; 75 ng). After stimulation with Wnt3a-CM or Co-CM (20
hrs), cells were lysed and subjected to luciferase measurements or in parallel to WB
analysis. All data represent mean +/- SD from at least 4 independent experiments performed
in triplicates. Statistical significance was analyzed using the student´s t-test (*p< 0.05).
Fig. S4 CD44 augmentation of Wnt/β-catenin signaling is independent of RTK
signaling via the MEK/Erk- and PI3K/Akt-pathways.
A) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together
with an empty vector or a cDNA for hCD44s (75 ng), treated either with DMSO or the MEKInhibitor U0126 (15 µM) 1 h prior to the addition of Co-CM or Wnt3a-CM. 20 hrs after
induction, cells were lysed and subjected to luciferase measurements. B) HEK293 cells were
transfected similar to A), treated either with DMSO or the PI3K-Inhibitor Ly294002 (50 µM) 1
h prior to the addition of Co-CM or Wnt3a-CM. 20 hrs after induction, cells were lysed and
subjected to luciferase measurements. Reporter gene assays are described in “Materials and
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Methods”. All data represent mean +/- SD from at least 4 independent experiments
performed in triplicates. Statistical significance was analyzed using the student´s t-test (*p<
0.05).
Fig. S5 The complex between CD44 and LRP6 occurs through the cytoplasmic
domains and is dependent on the binding of CD44 to ERM proteins.
A) HeLa cells were transfected with either empty vector or FLAG-tagged LRP6. 24 hrs after
transfection cells were treated either with Co-CM or Wnt3a-CM for 30 minutes, lysed and
FLAG-LRP6 proteins were immunoprecipitated with a FLAG-antibody. Immunoprecipitates
were subjected to WB analysis with FLAG- and hCD44 antibodies. B) HeLa cells were
transfected with cDNAs for CD44s or CD44s mutated in the ERM binding domain (CD44sERMmut) together with a FLAG-tagged LRP6 construct. 24 hrs after transfection cells were
treated with Wnt3a-CM for 30 minutes, lysed and proteins were immunoprecipitated with an
antibody against CD44. Immunoprecipitates were subjected to WB analysis using FLAG- and
CD44 antibodies. C) HeLa cells were transfected with CD44-Cyt-GST or FLAG-LRP6∆E(1-4)
alone or with CD44-Cyt-GST together with either empty vector or FLAG-LRP6∆E(1-4). 48 hrs
later cells were lysed and lysates were subjected to GST-pulldown as described in “Materials
and Methods”. The precipitates were subjected to WB analysis using antibodies against
FLAG and GST.
Fig.S6 xCD44-MO injected embryos show a musculature defect phenotype that can be
rescued by hCD44s.
Xenopus laevis embryos used for the in situ hybridization (Fig. 7b) were analyzed
morphologically. Embryos were injected in the right blastomere at 2-cell stage either with
Control-MO, xCD44-MO alone or xCD44-MO together with hCD44s expression vectors. FITC
labeled Dextran was co-injected to assess the side of injection. Numbers indicate the amount
of analyzed embryos. Percentage of embryos with musculature defects (indicated by an
arrow) was quantified (histogram). Data represent mean +/- SD from at least 4 independent
experiments. Numbers indicate the amount of analyzed embryos. Statistical significance was
analyzed by the student´s t-test (*p< 0.05).
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