Supplementary Figure 1. SPRY2 Is Not a B-Catenin/TCF Target Gene. (A ) Graph Representing

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 . 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 (FOP- flash). 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, non- differentiated 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

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 upper 16 patients were scored as high and the lower 14 as low SPRY2 expressing. The cut-

3 off was the median value obtained for the relative SPRY2 expression in all 30 patients analysed.