Supplementary Figure 1. SPRY2 is not a -catenin/TCF target gene. (a) Graph
representing -catenin enrichment in the two TCF/LEF binding sites (A and B) described for
the human LEF1 gene (48). All values correspond to specific -catenin enrichment versus
precipitation with control IgG and amplification of an unrelated downstream control region
(dsCtrl). (b) Graph representing the Luciferase activity under the control of four TCF
response elements (TOP-flash) in LS174T-tetON-NTCF4 colon cancer cells treated as
indicated. Values were corrected by the activity of a mutant version of the luciferase
construct in which the response elements can not bind TCF transcription factors (FOPflash). Bars correspond to the mean  SD of three independent experiments. (c) Total
extracts of LS174T-tetON-NTCF4 colon cancer cells were analysed by Western blot to
detect the levels of C-MYC, AXIN2 and SPRY2 proteins upon treatment with DOX. Fold
change of DOX-treated versus non treated cells is indicated for the amount of both proteins
and corrected by -actin expression.
Supplementary Figure 2. SPRY2 is expressed in cells at the proliferative, nondifferentiated and Wnt active region at the bottom of the colon crypts. (a-c)
Representative pictures showing protein expression by double immunofluorescence and
confocal microscopy of (a) SPRY2 (green) and CD44 (red), (b) PanCytokeratin (green) and
SPRY2 (red) or (c) Ki67 (green) and SPRY2 (red). (a). Insets below show magnifications
corresponding to an area at the middle (1) or at the bottom (2) of the crypt that are delineated
by dashed squares on the main picture. Asterisks indicate SPRY2 positive staining in
stromal cells. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 50 m. (d)
Diagram representing the spatially-organized cell expression of SPRY2, nuclear FOXO3a
and -catenin in coordination with Wnt/-catenin pathway activity, cell proliferation and
differentiation along the self-renewing human colon crypts.
1
Supplementary figure 3. Spry2 expression in mouse epidermis correspond to cells that
accumulate nuclear FOXO3a and -catenin in growing anagen hair follicles. (a)
Representative pictures showing protein expression by double immunofluorescence and
confocal microscopy of FOXO3a (red) and -catenin (green). Thick dashed lines mark hair
matrix region (M). Insets below show magnifications corresponding to an area delineated by
a dashed square on the main picture. Dashed lines in insets mark four nuclei with the highest
double -catenin and FOXO3a co-localization signal (yellow). (b) Left panel, image
representing nuclear -catenin and FOXO3a co-localization at the region shown in the inset
of panel a. Dashed lines mark nuclei with double staining for -catenin and FOXO3a. Right
panel, colour scatter plot showing the corresponding intensity correlation analysis of green
(-catenin) and red (FOXO3a) channels of the nuclei mark by dashed lines in panel b.
Yellow dots represent co-localization signal. (c) Quantification by qRT-PCR of a ChIP
assay to detect the enrichment of -catenin at the DBE sites (B and C) described at the
proximal region of the mouse Spry2 gene promoter (Figure 1d). The ChIP was performed on
an extract obtained from mouse back skin rich in growing anagen hair follicles
(Supplementary Figure 4e). Mean ± SD of three independent experiments. P value
corresponds to non-parametric t test (d, e) Representative pictures showing protein
expression by double immunofluorescence and confocal microscopy of (d) -catenin (green)
and Spry2 (red) or (e) FOXO3a (green) and Spry2 (red). Insets below show magnifications
corresponding to an area delineated by a dashed square on the main picture. Dashed lines in
the insets mark the nuclei of cells that express cytoplasmatic Spry2 and nuclear (d) -catenin
or (e) FOXO3a in the hair matrix. (a, d, e) Merge signal from green and red channels is
shown in yellow. Nuclei were stained with Hoechst 33342 (blue). (a, b, d, e) Scale bars, 100
m.
2
Supplementary Figure 4. Spry2 is expressed in Wnt-activated cells at the matrix of
anagen hair follicles. Representative pictures showing protein expression by double
immunofluorescence and confocal microscopy of (a) Spry2 (green) and Lef1 (red), (b)
Spry2 (green) and CD44 (red) or (c) Ha1 (green) and Spry2 (red). (a-c) Merge signal from
green and red channels is shown in yellow. Nuclei were stained with Hoechst 33342 (blue).
(d) Haematoxylin and Eosin staining of a mouse anagen hair follicle. In this phase, hair
follicles are stimulated by Wnt ligands secreted by dermal papilla cells (DP) that promote
accumulation of nuclear -catenin and proliferation of adjacent epithelial hair follicle matrix
cells (red line). A sub-population of cells within this Wnt/-catenin activated domain, also
accumulate nuclear FOXO3a (white line). Spry2 positive cells correspond to those that
simultaneously accumulate nuclear -catenin and FOXO3a and their immediate offspring
(dashed green line). (h) Haematoxylin and Eosin staining of mouse back skin 30 days after
shaving. (a-e) Scale bars, 100 m.
Supplementary Figure 5. SPRY2 expression and Pan-cytokeratin levels in human colon
cancer. The expression of SPRY2 and cytokeratins (PanCK) was analysed by
immunofluorescence and confocal microscopy in serial tissue sections of those same
patients shown in Figure 5. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 100
µm.
Supplementary table 1. Clinico-pathological information of 30 stage III CRC patients
analysed for nuclear FOXO3a/-catenin, and SPRY2 content. Age, sex, lymph nodes
affected (L. N.), tumor grade (G1-3), overall survival (months) and tumor expression of
SPRY2 and nuclear FOXO3a/-catenin are indicated for the 30 CRC patients studied. The
3
upper 16 patients were scored as high and the lower 14 as low SPRY2 expressing. The cutoff was the median value obtained for the relative SPRY2 expression in all 30 patients
analysed.
4