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Supplementary Figure Legends
Supplementary Figure S1 Expression of EpCAM, LEF1 in human endometrial cancer patients,
cancer cell lines and mouse endometrial tissues. A, Immunohistochemistry staining of epcam
and lef1 in mouse normal and tumor endometrial tissues. B, TCGA gene expression of EpCAM
and LEF1 in normal and endometrial carcinoma patients. *** if P<0.005, **** if P<0.001.
Supplementary Figure S2 Quantification of EpEX immunofluorescence staining and EGFR
expression in endometrial cancer cell lines. Reduced migration in EpCAM-edited RL95-2 cells.
A, Quantification of EpEX immunofluorescence staining intensity in EGF, Iressa, and DAPTtreated RL95-2 and Ishikawa cells. In EGF-treated RL95-2 cells showed reduced CTCF
(corrected total cell fluorescence) value (1.45 x 105) compared to control cells (CTCF: 1.35 x
106). B, EGFR expression in RL95-2, Ishikawa and AN3CA. C, Immunofluorescence staining of
EpEX in EGF treated AN3CA endometrial cancer cells. * if P<0.05. D, EpCAM-edited cells
exhibit reduced migration, but no significant changes in cell proliferation. The results were
determined as means  SD from 3 independent experiments. **p<0.01, ****p<0.001.
Supplementary Figure S3 Summary of ChIP-seq of EpICD in RL95-2 cells. A, Overview of
numbers of ChIP-seq peaks and false discovery rate (FDR) in each EGF treatment. B, Number
of genome-wide distribution of EpICD-ChIP-seq target loci. C, Pathway enrichment analysis of
EpICD-TSS-binding genes using KEGG database.
Supplementary Figure S4 Overview of ChIP-seq of LEF1 in RL95-2 cells. A, Number of
genome-wide distribution of LEF1-ChIP-seq target loci. B, Pathway enrichment analysis of
LEF1-TSS-binding genes using KEGG database.
Supplementary Figure S5 Pathway enrichment analysis unique/shared TSS-bound genes in 12
and 24 hours EGF. Top, Pathway analysis of unique EpICD/LEF1 ChIP-seq target loci. Bottom,
Pathway enrichment analysis of EpICD-LEF1-shared-TSS-binding genes using KEGG
database.
Supplementary Figure S6 105 EpICD-LEF1-TSS gene expression profiles in UCEC (Uterine
Corpus Endometrial Carcinoma) patients and normal control samples in TCGA cohort. A,
Unbiased hierarchical clustering of 105 gene expression showed 5 distinct groups in tumor
patients. The distance between normal control and 5 tumor groups represented the degree of
similarity in their expression profiles.
In particular, group 5 showed the most different
expression profile compared to normal while group 1 patients shared the most similarity with
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normal control. However, some genes (highlight in grey boxes) showed the altered expression
profiles in group 1 and normal control, and these genes may have different implications in
promoting tumor progression. B, The composition of clinical stages in 5 tumor groups. Expect
for group 1, the rest of 4 groups of tumor patients showed an increase of percentages of
advanced stages as their expression profiles of 105 genes differ farther from normal control.
This suggested the expressions of these genes may correlate with tumor progression.
Supplementary Figure S7 EpCAM-edited cells showed reduced EGF-stimulated gene
activation. A, RT-qPCR on GNA11 and TGFA in RL95-2, EpCAM edited Clone 1 and Clone 2
cells in EGF treatment. B, RT-qPCR on co-regulated gene in RL95-2 and EpCAM edited Clone
1 cells in EGF treatment. The results were determined as means  SD from 3 independent
experiments. *** if P < 0.005, ** of P < 0.01, * if P < 0.05.
Supplementary Figure S8 RL95-2 cells showed no apoptosis and cytoskeleton content
induced by EGF treatments in RL95-2 and EpCAM-edited clone. A, Cell apoptosis with different
EGF time point were measured by Alexa Fluor 488 Annexin V/Dead Cell kit. B, Cytoskeleton
content was determined by immunofluorescence staining of α-tubulin and actin in RL95-2 and
EpCAM-edited cells. (Bar size=10 µm).
Supplementary Figure S9 EpCAM molecules were detected with recognition AFM only on cells
expressing this protein. A, Force plots illustrating interactions between an AFM tip decorated
with anti-EpCAM antibodies and EpCAM molecules on an RL95-2 cell. Each recognition event
is detected as a “jump” in force plots resulting from extra force needed for separation of the
antibody from the EpCAM molecule. The force involved in separation is in order of 100 pN and
the event occurs after pulling a long linker and likely some cell membrane. Unspecific binding,
performed with the tip decorated with antibodies specific to non-membrane protein Rpt5, shows
only shallow jumps superimposed on a broad adhesion depression. The control trace, collected
with a HEK293T cell not expressing EpCAM, does not detect any adhesive interactions. B,
Presence of EpCAM molecules was detected on EpCAM expressing hESC cells with probes
modified with anti-EpCAM antibodies but not with the standard probe. Size of peak force error
and adhesion images is 10x10mm; adhesion event maps are 3x3mm. C, Un-binding force
distribution of adhesion events between RL95-2 cells and AFM tip: (a) Decorated with anti
hEpCAM monoclonal antibodies; (b) As in (a) but cells were pre-incubated with anti hEpCAM
monoclonal antibodies; (c) Decorated with goat IgG; (d) Unlabeled. Adhesion events detected
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on force plots were quantified based on the height of the probe “jumps” and sorted into arbitrary
bins. Events measured below 10 pN were at the level of instrumental noise. Rupture between
membrane bound EpCAM and the bound antibody occurred at forces larger than 50 pN. After
the blocking membrane EpCAM molecules with the specific antibodies such events became
substantially less frequent, and the strongest events were not detected. The IgG decorated tips
only weakly interacted with the surface proteins. A tip without any chemical decoration sensed
only events at a level of the instrumental noise. D, Texture parameters calculated from adhesion
measurements performed with an anti-EpCAM antibodies decorated probe on RL95-2 cells. E,
Roughness analysis of recognition events confirms gradual disappearance of EpCAM
molecules from a RL95-2 cell surface and detects formation of EpCAM islands. Root mean
square (RMS) of adhesion and the maximal relative adhesion systematically decreased with the
EGF exposure time. Events density dropped abruptly after the 12-hr exposure but kurtosis
increased indicating a more pointed surface. * if P < 0.05.
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