Quantitative RT-PCR primersGene Genebank Forward Primer Reverse PrimerEpCAM NM_002354 AATGTGTGTGCGTGGGA TTCAAGATTGGTAAAGCCAGTOct-4 NM_002701 GGAGAGCAACTCCGATGG TTGATGTCCTGGGACTCCTCCOL3A1 NM_000090 TCCGGGTGAGAAAGGTGA GCAGGTCCAGAACCTCCAGc-Myc NM_002467 AAACACAAACTTGAACAGCTAC ATTTGAGGCAGTTTACATTATGGNanog NM_024865 ATGCCTCACACGGAGACTGT AGGGCTGTCCTGAATAAGCASox2 NM_003106 TATTTGAATCAGTCTGCCGAG ATGTACCTGTTATAAGGATGATATTAGTKlf4 NM_004235 ACCAGGCACTACCGTAAACACA GGTCCGACCTGGAAAATGCTGAPDH NM_002046 CTTCACCACCATGGAGGAGGC GGCATGGACTGTGGTCATGAG

ChIP primersGene Genebank Forward Primer Reverse PrimerEpCAM (-630 to -550) NM_002354 ACATCTTCAAGTGCTAGAAATGC GAAATCTTGGCTCTCTTGGGEpCAM (-354 to -273) NM_002354 CCATTCTTCAAGGCTTCAGAG GGCGTTAGGGATCTTTGGTEpCAM (+426 to +539) NM_002354 CCTCACTTCGCAGCTTTG GCCGCAGGAAACCTGGAEpCAM (+835 to +967) NM_002354 GCTTATTGTAGGGAACGCAG CGACAGAGCAAGACTCAGc-Myc (-341 to -208) NM_002467 GCCTGCGATGATTTATACTCAC AAACAGAGTAAGAGAGCCGc-Myc (+214 to +358) NM_002467 CTAGGGTGGAAGAGCCG GCTGCTATGGGCAAAGTTOct-4 (-173 to -96) NM_002701 CAGTTGTGTCTCCCGGTTTT CAGTTGTGTCTCCCGGTTTTOct-4 (-2613 to -2396) NM_002701 GGGGAACCTGGAGGATGGCAAGCTGAGAAA GGCCTGGTGGGGGTGGGAGGAACATKRT1 NM_006121 GTGGCACAGAGTTTAGTGA TGGGTACTCTTGCACTTGNanog NM_024865 TCTTCAGGTTCTGTTGCTCG GTTAATCCCGTCTACCAGTCTCSox2 NM_003106 GGATAACATTGTACTGGGAAGGGACA CAAAGTTTCTTTTATTCGTATGTGTGAGCAKlf4 NM_004235 GGAAAGGAGAGTGCGTG CACTGCCTGTAATATTTGATGACTAAHBB NM_000518 ATCTGAGCCAAGTAGAAGACCTTTTC TCTGCCTGGACTAATCTGCAAGGAPDH NM_002046 TCCAAGCGTGTAAGGGT GAAGGGACTGAGATTGGC

MSP primersGene Genebank Forward Primer Reverse PrimerEpCAM-Methylated NM_002354 TTTAACGTCGTTATGGAGACGA GCTAATACTCGTTAATAAATCACCGEpCAM-Unmethylated NM_002354 TTTAATGTTGTTATGGAGATGA ACCACTAATACTCATTAATAAATCACCAC

Bisulfite sequencing primersGene Genebank Forward Primer Reverse PrimerEpCAM NM_002354 AAGGAAGTTTTAGTATAGAATTTTTAAAT AAAAAAATAAATAAACTCCCCTCCOct-4 NM_002701 ATTTGTTTTTTGGGTAGTTAAAGGT CCAACTATCTTCATCTTAATAACATCC

TABLE S1 – Primers Used for Cloning, Quantitative PCR/RT-PCR

100 101 102 103 1040

11

23

34

45

100 101 102 103 1040

9

18

26

35

100 101 102 103 1040

11

23

34

45

100 101 102 103 1040

14

28

41

55

100 101 102 103 1040

11

23

34

45

H9

H9-Diff. hES5-Diff.

hES5 HUES6

HUES6-Diff.

Fluorescence intensity

Cel

l cou

nt

Fig. S1. Confirmation of undifferentiated and differentiated hESCs by measuring cell surface SSEA4 expression. Cell surface SSEA4 protein expression by undifferentiated (H9, hES5 and HUES6) and differentiated (H9-Diff., hES5-Diff. and HUES6-Diff.) hESCs were investigated by flow cytometric analysis using an anti-SSEA4 MAb. Fluorescence minus control without addition of the anti-SSEA4 antibody was used as a negative control (grey histogram).

H9

50μm 5μm25μm

50μm 5μm25μm

H9-Diff.

Fig. S2. Nuclear localization of EpICD in undifferentiated hESCs. Undifferentiated H9 and H9-Diff. cells were probed with EpICD-specific antibodies (1144-1) in combination with rhodamine-labeled secondary antibodies. Localization of EpICD(red) was assessed with laser scanning confocal microscopy. Nuclei were counterstained with DAPI (blue).

0

0 .5

1

1 .5

2

2 .5Se

coda

ryAb

Cont

rol R

abbi

t IgG

Rabb

it an

ti-Ep

CAM

MAb

(114

4-1)

Cont

rol G

oat I

gG

Goat

ant

i-EpC

AMse

ra (A

-20)

OD

490n

m

Fig. S3. Characterization of antibodies’ specificity against synthetic human EpICD peptide. ELISA analyzing binding affinity of the two commercial available anti-EpCAM Abs (1144-1 and A-20) against human EpICD was performed. First antibodies (5 μg/ml) were added onto synthetic EpICD peptide-coated ELISA plates. Secondary antibodies only were used as a non-specific binding control. Both the two anti-EpCAM Abs (1144-1 and A-20) showed highly specific binding affinity to the synthetic human EpICD peptide.

0

0 .2

0 .4

0 .6

0 .8

1

1 .2

■ EpCAM

mR

NA

exp

ress

ion

leve

ls

0

0 .2

0 .4

0 .6

0 .8

1

1 .2

■ Oct-4

Mock

control shRNAshRNA1

shRNA2Mock

control shRNAshRNA1

shRNA20

0 .2

0 .4

0 .6

0 .8

1

1 .2

■ Nanog

Mock

control shRNAshRNA1

shRNA2

0

0 .2

0 .4

0 .6

0 .8

1

1 .2

■ Sox2

mR

NA

exp

ress

ion

leve

ls

Mock

control shRNAshRNA1

shRNA20

0 .2

0 .4

0 .6

0 .8

1

1 .2

■ Klf4

Mock

control shRNAshRNA1

shRNA2

Fig. S4. Knocking down the expression of EpCAM suppressed Oct-4, Nanog, Sox2 and Klf4 expression in HCT116. Cells treated with EpCAM shRNAlentivirus caused knockdown of EpCAM expression (~66%), and downregulatedthe expression of Oct-4 (~68%), Nanog (~76%), Sox2 (~55%), and Klf4 (~36%) mRNA expression, compared to control mock cells. Total RNAs derived from HCT116 mock, HCT116/control shRNA, HCT116/shRNA1 and HCT116/shRNA2 cells were quantitated by Q-RT-PCR and normalized to GAPDH levels. To obtain relative expression levels, genes’ expression in mock cells was set at 1.0.