Fig. S1. Effect of p120-catenin overexpression on the interaction of SCUBE2 with E-cadherin. The expression plasmid encoding FLAG.

Transcription

1 Fig. S1. Effect of p120-catenin overexpression on the interaction of SCUBE2 with E-cadherin. The expression plasmid encoding FLAG.SCUBE2, E-cadherin.Myc, or HA.p120-catenin was transfected in a combination as indicated into HEK-293T cells. After 2 days, cell lysates underwent immunoprecipitation (IP), then western blot (WB) analysis with the indicated antibodies to determine the protein protein interactions.

2 Fig. S2. Pulldown assays with recombinant SCUBE2 protein and a GST-fusion protein containing the juxtamembrane region of the E-cadherin cytoplasmic domain. (A) Production of a GST-fusion protein containing the p120-catenin binding site (amino acids ) within the E-cadherin cytoplasmic domain. Schematic representation of E-cadherin and the GST-fusion construct containing the p120-catenin binding site (GST.E-cadherin ). (B,C) Pull-down assays. Recombinant HA epitope-tagged p120- catenin (HA.p120-catenin) or FLAG.SCUBE2 proteins (FL, ty97, or D4) was produced by overexpression from HEK-293T cells. Purified GST.E-cadherin protein was mixed with recombinant HA.p120-catenin protein bound to anti-ha antibody agarose beads as a positive control (B) or recombinant FLAG.SCUBE2-FL, ty97, or D4 protein bound to anti-flag antibody agarose beads (C). After incubation for 4 h at 4 C, the beads were washed extensively, and the interacting protein was visualized by immunoblotting with anti-gst antibody. Note that p120-catenin (B) but none of SCUBE2 protein fragment (C) could directly interact with the juxtamembrane region of the E-cadherin cytoplasmic domain.

3 Fig. S3. SCUBE2 expression is associated with E-cadherin but inversely correlated with the migratory and invasive abilities of breast-cancer cell lines. (A) RT-PCR analysis of mrna expression of SCUBE2 and E-cadherin in MCF-7, T-47D, MDA-MB-231, and ZR cells. GAPDH was a normalization control and lack of cdna a negative control (-). (B) Western blot (WB) analysis of protein levels of SCUBE2 and E-cadherin. b-actin was a loading control. (C) Morphologic features of each breast-cancer cell lines. Images were taken under phase-contrast microscopy. Bar, 50 um. Invasion (D,F) and migration (E,G) assays of these breast-cancer cell lines. Breast-cancer cells were seeded on Transwells coated with 30 mg Matrigel. After 18-h incubation, the invaded cells were fixed, stained, and photographed. Representative photographs of Transwell membranes showing invaded cells (D) stained with crystal violet, and OD 570 was measured to quantify the amount of DMSO-solubilized dye. All assays were performed in triplicate. Quantitative data are mean ± SD. (F). Spontaneous motility of the 4 breast-cancer cell lines examined over 12 h in confluent monolayers wounded by a cell culture insert. The wound was photographed at 0 and 12 h after the scratch (E), and the mean±sd proportion of scratch wound closure was calculated (G).

4 Fig. S4. Effect of TGF-b1 treatment on apoptosis and EMT in MCF-7 cells. (A,B) TUNEL assay. MCF-7 vector or SCUBE2- expressing cells before or after TGF-b1 treatment (21 d) underwent TUNEL or DPAI (nuclear) staining. Representative images (A) or quantitative analysis of TUNEL-positive cells (B) from each experimental group are shown. Data are mean±sd from 3 experiments. (C) Active caspase-3 protein expression. MCF-7 Tet-off vector or FLAG.SCUBE2-expressing cells were treated with TGF-b1 (10 ng/ ml) for 7, 14, and 21 days. Cell lysates derived from these cells were probed with a specific antibody against the cleaved, active form of caspase-3 (17 and 19 kda). WB, western blot analysis. (D) TGF-b1-induced EMT resulted in cell morphological changes. MCF-7 cells were treated with TGF-b1 (10 ng/ml) for 7, 14, and 21 days. Cell morphologic alterations associated with EMT are shown in phase-contrast images. Bars, 50 mm. MCF-7 cells were treated with TGF-b1 at various concentrations for 3 days (E) or with TGF-b1 (10 ng/ml) for the indicated times (F). Western blot analysis of protein levels of SCUBE2, E-cadherin, vimentin, or b-actin.

5 Fig. S5. SCUBE2 overexpression promoted cell cell aggregation in a Ca 2+ -dependent manner. (A) Degree of aggregation in suspension of MDA-MB-231 control and SCUBE2-expressing cells. MDA-MB-231 control or SCUBE2-expressing cells were detached and allowed to aggregate in suspension culture in the presence (+) or absence (-) of Ca 2+ in aggregation medium for 9 h. Aggregates were defined as clusters of 4 or more cells. Data are mean±sd of 3 experiments. *, p<0.01 versus control cells. (B) Representative micrographs of aggregates formed by MDA-MB-231 control or SCUBE2-expressing cells. The experiments were performed 3 times in duplicate with similar results. Original magnification, 3100.

6 Fig. S6. SCUBE2-E-cadherin interaction is critical for the SCUBE2-driven epithelial transition and FOXA1 upregulation in MDA-MB-231 cells. (A) Domain organization of the SCUBE2 expression constructs (FL, ty97, and D4) (see Fig. 1B). Graphic illustration for the locations of the oligonucleotide primer pairs used to amplify the NH 2 (N-S2-f and r primers) or COOH-terminal (C-S2-f and r primers) region of SCUBE2 cdna sequence for FL, ty97, and D4 mutant. (B,C) Effects of SCUBE2-FL, ty97, and D4 on the expression of EMT marker genes or FOXA1 in MDA-MB-231 cells. Cells were transfected with the empty vector or the expression plasmids encoding SCUBE2-FL, ty97, or D4 mutant protein. Two days after transfection, the first-strand cdnas or protein lysates derived from these transfected cells were used to perform RT-PCR (B) or western blotting analysis (C) for the mrna or protein levels of SCUBE2, E-cadherin, vimentin, N-cadherin, and FOXA1 together with GAPDH, b-actin, or lamin A/C serving as an internal control.

7 Fig. S7. Classical EMT inducers suppress E-cadherin but not SCUBE2 in MCF-7 cells. (A-D) Effects of the E-cadherin repressors on SCUBE2 expression in MCF-7 cells. MCF-7 cells were transiently transfected with the empty vector or the expression plasmid encoding the E-cadherin repressors (TWIST, SNAIL, SLUG, ZEB1, or ZEB2) individually. Two days after transfection, transfected MCF-7 cells were collected and lysed to perform RT-PCR or western blot analyses for the expression of these E-cadherin repressors or SCUBE2 at both mrna (A,C) and protein levels (B,D). (E) Effects of the E-cadherin repressors on E-cadherin or SCUBE2 promoter activity. Expression plasmids encoding the E-cadherin repressors as indicated or the empty vector (250 ng) were co-transfected with 250 ng each of the empty reporter vector (pgl3), E-cadherin promoter (-995/+135) construct, or SCUBE2 promoter (-1425/+1) construct into MCF-7 (E-cadherin and SCUBE2-positive) cells in a 24-well plate. Two days later, firefly luciferase activity was analyzed relative to Renilla luciferase activity to control for transfection efficiency. Relative luciferase activity was further normalized to values obtained in the empty vector-transfected cells. Data are mean ± SD from 3 independent experiments.

8 Fig. S8. Restoration of SCUBE2- and E-cadherin-silenced expression by 5-aza-29-deoxycytidine (AZA). (A) Quantitative RT- PCR analysis of mrna level of SCUBE2 in MDA-MB-231 cells treated with AZA (10 mm), trichostatin A (TSA; 300 nm), or both, for 96 or 24 h. GAPDH level was a normalization control. Data are mean±sd of 3 experiments. *, P<0.01 compared with the control. (B) RT-PCR analysis of mrna level of E-cadherin in MDA-MB-231 cells treated with AZA (10 mm), TSA (300 nm), or both, for 96 or 24 h. GAPDH level was a normalization control. Lack of AZA and TSA treatment was a negative control. (C) Western blot (WB) analysis of protein level of SCUBE2 and E-cadherin in MDA-MB-231 cells treated with AZA (10 mm), TSA (300 nm), or both, for 96 or 24 h. b-actin level was a loading control. Lack of AZA and TSA treatment was a negative control.

9 Fig. S9. Effect of in vitro methylation on SCUBE2 gene reporter activity. (A) Schematic diagram mapping the methylated CpG dinucleotides within the upstream 1,500 bp of the promoter (Prom) and downstream to of SCUBE2, which covers exon 1 (Ex1), intron 1 (In1) and exon 2 (Ex2). Vertical lines represent CpG sites (upper panel). Genomic fragments (Promoter, Ex1+In1, and In1+Ex2) used to construct the luciferase reporter plasmids are marked (lower panel). (B) Methylation analysis of reporter constructs. Agarose gel electrophoresis of methylated (M, Sss I +) and unmethylated (UM, Sss I -) reporter plasmids of Prom-Luc, Ex1+In1-Luc and In1+Ex2-Luc digested with methylation-sensitive restriction endonuclease HpaII (does not cut methylated DNA). (C) Quantification of activity of the Prom-Luc, Ex1+In1-Luc and In1+Ex2-Luc constructs transiently transfected into MCF-7 cells. Luciferase activity was analyzed relative to Renilla luciferase activity (prl-tk) to control for transfection efficiency. Data are mean±sd of 3 experiments. *, P<0.01 compared with the control.

10 Fig. S10. The protein expression of DNMT1 in human breast-cancer cell lines and in vivo binding of DNMT1 protein to the exon 1 region of SCUBE2. (A) Western blot analysis of protein level of DNMT1 in MCF-7 and MDA-MB 231 cells. Nuclear lamin A/C level was a loading control. (B) Chromatin immunoprecipitation assay of in vivo binding of DNMT1 protein to CpG sites (+346 to +592) of SCUBE2 in MCF-7 and MDA-MD-231 cells analyzed by PCR with specific primers for SCUBE2 CpG sites (see Figure 8A). Primers amplified a 246-bp fragment. Lack of cell lysates (-) was a negative control.

11 Table S1. Primers used in this study Gene Forward Reverse Assay SCUBE2 CCCCCAAGCGCCGCATCCTGA TATTGAGTGGCACGTGGGCTGAGT RT-PCR N-SCUBE2 GCAGTTGCTGACTACAAAGACG CAGGCACTCGTCCACATCAA RT-PCR C-SCUBE2 CCCCCAAGCGCCGCATCCTGA TATTGAGTGGCACGTGGGCTGAGT RT-PCR E-cadherin AGTGCCAACTGGACCATTCA TCTTTGACCACCGCTCTCCT RT-PCR Vimentin CGTACGTCAGCAATATGAAAGTGT GTGTCTTGGTAGTTAGCAGCTTCA RT-PCR N-cadherin CACTGCTCAGGACCCAGAT TAAGCCGAGTGATGGTCC RT-PCR b-catenin CCTGTGCAGCTGGAATTCTTT ACAACTGGTAGTCCATAGTGA RT-PCR FOXA1 CGCTTCGCACAGGGCTGGAT TGCTGACCGGGACGGAGGAG RT-PCR TWIST GGAGTCCGCAGTCTTACGAG TCTGGAGGACCTGGTAGAGG RT-PCR SNAIL TGCGCTACTGCTCGGCGAAT AGGGCTGCTGGAAGGTAAACTCTGG RT-PCR SLUG CTGGGCGCCCTGAACATGCAT GGCTTCTCCCCCGTGTGAGTTCTA RT-PCR ZEB1 AGTGGTCATGATGAAAATGGAACACCA AGGTGTAACTGCACAGGGAGCA RT-PCR ZEB2 GACAGATCAGCACCAAATGC GCTGATGTGCGAACTGTAGG RT-PCR DNMT1 GAGGAAGCTGCTAAGGACTAGTTC ACTCCACAATTTGATCACTAAATC RT-PCR GAPDH GCCAAAAGGGTCATCATCTC ACCACCTGGTGCTCAGTGTA RT-PCR SCUBE2 (+159~+592) TTTGGTATAGTAGGTAGGGTTAGGAA ACAAAAAAATCTCTAAAACCCACCC EpiTYPER SCUBE2 (M) GTTAGTTAGATCGTCGTCGGC GAAACAAACGCTATTCCGCT MSP SCUBE2 (UM) TTGTTAGTTAGATTGTTGTTGGTGA CAAAACAAACACTATTCCACT MSP SCUBE2 (+346~+592) CCCGGAGCTGCTGACGGTTCCCGC ACAGAAAAGTCTCTGGAGCCCACC ChIP E-cadherin (-371~-279) CTCCAGCTTGGGTGAAAGAG GGCCTTTTACACTTGGCTGA ChIP M, methylated; UM, unmethylated; MSP, methylation-specific PCR; ChIP, chromatin immunoprecipitation. Table S1. Primers used in this study