Legends to supplementary figures
Supplementary Fig. 1 Western blot analysis for protein expression of E-cadherin and
Vimentin in a panel of OSCC cell lines and RT7.
Supplementary Fig. 2 COBRA for CpG-islands within/around EMT-related genes and candidate genes in 18 OSCC cell lines, Panc1 and H661 cell lines. PCR products used for
COBRA were digested with Bst UI or Taq I (primers, Supplementary Table 1), and electrophoresed (see the summary in Fig. 2B). Stars indicate samples in which DNA hypermethylation of CpG-islands was detected by COBRA.
Supplementary Fig. 3 Bisulfite sequencing of representative OSCC cell lines with
(epithelial-like cell line, E) or without (mesenchymal-like cell line, M) the expression of candidates in regions within/around these genes. Maps of CpG sites, and PCR products used for COBRA and bisulfite sequencing (primers, Supplementary Table 1; see Fig. 2 and
Supplementary Fig. 2) are indicated above the results of bisulfite sequencing. Vertical tick marks , CpG sites; horizontal bars with arrowheads , PCR products; and vertical arrows , restriction enzyme sites. Open and filled squares represent unmethylated and methylated
CpG sites respectively, and each row represents a single clone.
Supplementary Fig. 4 MSP analysis of miR-200b/a , WNT7A , and WNT10A in a panel of
OSCC cell lines ( A ) and primary OSCCs ( B ). PCR products (primers, Supplementary Table
1) were electrophoresed. M, methylated allele; U, unmethylated allele. Stars indicate hypermethylation-positive samples. OSCC cell lines in which DNA hypermethylation of candidate genes was detected by COBRA (see Supplementary Fig. 2 and the summary in Fig.
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2B) were used as positive and negative controls. The correlation between clinical background and results of the MSP analysis is shown in Table 1-3.
Supplementary Fig. 5 Analyses of phenotypic plasticity of an epithelial-like OSCC cell line, OM1, and two mesenchymal-like OSCC cell lines, HOC313 and TSU. A , Effects of dsRNA mimicking miR-200a , or miR-200b or control non-specific miRNA on expression of
E-cadherin , WNT7A , and WNT10A in mesenchymal-like OSCC cell lines HOC313 and TSU.
NT indicates not treated. In RT-PCR analysis, expression levels of these genes72 hours after transfection were based on the amount of target message relative to the GAPDH transcript to normalize the initial input of total RNA. Bar graphs show the ratio of the expression in dsRNA-transfectants to that in RT7 cells cultured in KGM-2 BulletKit (Cambrex,
Walkersville, MD) supplemented with 25% DMEM and 2.5% fetal bovine serum for 7 days.
B , Effects of ZEB1 -overexpression on expression of E-cadherin and WNT7A in an epithelial
-like OSCC cell lines OM1. TaqMan real-time RT-PCR analysis for ZEB1 , E-cadherin , and
WNT7A ( left panel ) and Western blot analysis for E-cadherin ( right panel ) in OM1 cells 72 hours after transient transfection with a FLAG-tagged construct containing ZEB1
(pIRES-hrGFP IIZEB1 ) or empty vector (pIRES-hrGFP IIEmpty ) as a control. Expression levels of ZEB1 , E-cadherin , and WNT7A were based on the amount of target message relative to the GAPDH transcript to normalize the initial input of total RNA. Bar graphs show the ratio of expression levels in ZEB1 -transfectants to the control counterparts. Protein levels of
ZEB1 and E-cadherin in these transfectants were analyzed by Western blotting.
Supplementary Fig. 6 Expression levels of ZEB1 and ZEB2 in 18 OSCC cell lines, Panc1, and H661 cell lines. Expression levels of ZEB1 and ZEB2 were based on the amount of target message relative to the GAPDH transcript to normalize the initial input of total RNA.
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Bar graphs show the ratio of expression levels in these cell lines to a control counterpart RT7.
Supplementary Fig. 7 Effects of rhWNT7A on E-cadherin expression in Panc1 cells.
Western blot analysis for E-cadherin expression in Panc1 cells treated with 25ng/ml ( A ), with
50ng/ml ( B ), or without (NC) rhWNT7A in DMEM supplemented with 10% or 0.5% fetal bovine serum for 24, 48, and 72 hours.
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