The binding of p120ctn isoforms 1A and 3A with Kaiso.

<p>We introduced plasmids encoding DDK-MYC tagged p120ctn isoforms 1A and 3A cDNA into the lung cancer cell lines, and verified the effect of transfection by Western blot using p120ctn and MYC specific antibodies. P120ctn specific bands were detected at 120 kDa and 100 kDa and MYC specific bands were detected at 62 kDa in following transfection of the SPC (A) and LTE (E) cell lines. Statistical analysis by t-test in SPC (B) showed that cells transfection with p120ctn-1A (<i>P</i><0.001 for 24 h, <i>P</i><0.001 for 48 h, and <i>P</i> = 0.007 for 72 h, respectively) and -3A (<i>P</i> = 0.008 for 24 h, <i>P</i> = 0.001 for 48 h, and <i>P</i> = 0.008 for 72 h, respectively) showed significant expression, compared with control cells. Similar results to those in the SPC cell line were seen in the LTE line (F: in the cells transfection with p120ctn-1A, <i>P</i> = 0.013 for 24 h, <i>P</i> = 0.023 for 48 h, and <i>P</i> = 0.001 for 72 h, respectively; in the cells transfection with p120ctn-3A, <i>P</i><0.001 for 24 h, <i>P</i> = 0.002 for 48 h, and <i>P</i><0.001 for 72 h, respectively). Co-immunoprecipitation results confirmed that Kaiso formed complexes with proteins expressed by p120ctn isoform plasmid transfected SPC (C) and LTE (G) cell lines, and the statistical analysis by t-test in SPC (D) and LTE (H) showed that the binding ability of kaiso with p120ctn isoform 1A was significantly less than that of p120ctn isoform 3A (D: <i>P</i><0.001 for p120ctn, <i>P</i> = 0.002 for MYC, respectively; H: <i>P</i><0.001 for p120ctn, <i>P</i><0.001 for MYC, respectively).</p

Methylation status of the β-catenin promoter region in SPC.

<p>Mapping of the BSP results of lung cancer cell lines SPC, showing the methylation status of the β-catenin promoter region. The filled circles represent methylated CG sites, hollow circles represent unmethylated CG sites. Yellow indicates methylation, blue indicates unmethylated, gray indicates no CG site.</p

β-catenin promoter-specific primers for bisulfite (BSP) sequencing.

<p>β-catenin promoter-specific primers for bisulfite (BSP) sequencing.</p

P120ctn isoform 1A restored the cytoplasmic E-cadherin levels and enhanced the invasiveness in SPC cells.

<p>SPC cells were transiently transfected with GFP-siRNA-p120ctn or with empty vector as control. 24 hours after transfection, an aliquot of cells was transfected again with p120ctn isoform 1A or 3A cDNA plasmids. (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal shown in the nucleus and cytoplasm indicates effective expression of GFP from GFP-siRNA-P120ctn, confirming the successful transfection. Depletion of p120ctn (I) reduced the E-cadherin levels (II). Transfection with empty vector (III) did not affect the E-cadherin levels (IV). Restitution of p120ctn isoform 1A (V) restored the cytoplasmic E-cadherin levels (VI), while restitution of p120ctn isoform 3A (VII) had no effects on the E-cadherin levels (VIII). (<b>B</b>) Levels of E-cadherin were then analyzed by Western blot assay. The results confirmed that depletion of p120ctn resulted in decreased E-cadherin levels. Restitution of p120ctn isoform 1A restored the E-cadherin levels, while restitution of p120ctn isoform 3A had no effects on E-cadherin expression. (<b>C</b>) The invasiveness of SPC cells were analyzed by Matrigel invasion assay. P120ctn ablation reduced the cell invasiveness in comparison with the control group transfected with vector alone (*, <i>P</i><0.01). Restitution of p120ctn isoform 1A and 3A both enhanced the invasiveness of SPC cells in comparison with the group with p120ctn ablation (Si-p120ctn) (**, <i>P</i><0.01, ***, <i>P</i><0.01).</p

P120ctn isoform 1A, isoform 3A and five p120ctn 1A deletion mutants M1 to M5.

<p>P120 isoform 1A contains the coiled-coil domain and a central armadillo domain. P120 isoform 3A lacks the coiled-coil domain. Five p120ctn 1A deletion mutants M1 to M5 fused to green fluorescent protein (GFP): M1 contains only N-terminal 1–101 amino acids; M2 has N-terminal 1–54 amino acids deleted; M3 contains only N-terminal 1–54 amino acids; M4 has N-terminal 55–101 amino acids deleted; M5 contains only N-terminal 55–101 amino acids.</p

Mutant 4 restored cytoplasmic E-cadherin and enhanced the invasiveness in SPC cells.

<p>SPC cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn or M1–M5-GFP was expressed in the nucleus and cytoplasm. Expression (repletion) of M4 mutant (IX) restored cytoplasmic E-cadherin (X), while expression of the other mutants had no significant effects on the E-cadherin levels. (<b>B</b>) The levels of E-cadherin were analyzed by Western blot assay. Expression of M1–M5 mutants was detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated E-cadherin levels, whereas expression of the other mutants had no effects on the E-cadherin levels. (<b>C</b>) The invasiveness of SPC cells was analyzed by Matrigel invasion assay. Expression of M2 or M4 mutants enhanced the cell invasiveness in comparison with the group with ablated p120ctn (Si-p120ctn) (*, <i>P</i><0.01, **, <i>P</i><0.01), while expression of other three p120 1A mutants did not show significant effects.</p

Kaiso suppresses β-catenin mRNA expression in cell lines that have not been treated with 5-Aza-CdR.

<p>Lung cancer cell lines SPC (A) and LTE (E) were transfected with Kaiso cDNA plasmid. The effect of transfection at different time points was identified by Western blot. The Kaiso-specific band appears at 97 kDa. Statistical analysis of SPC (B) or LTE (F) by t-test showed a significant increase Kaiso expression in the cells transfected with Kaiso cDNA plasmid compared with the control cells (B: <i>P</i> = 0.003 for 24 h, <i>P</i> = 0.005 for 48 h, and <i>P</i><0.001 for 96 h, respectively; F: <i>P</i> = 0.065 for 24 h, <i>P</i> = 0.007 for 48 h; <i>P</i> = 0.009 for 96 h, respectively). Analysis of β-catenin mRNA expression in SPC following treatment of using RT-PCR (C) and Real-Time PCR by ANOVA (D) showed that treatment with 5-Aza-CdR demethylation reagent (7 µmol/L) for 48 h resulted in significant upregulation (<i>P</i> = 0.007), whereas high Kaiso expression significantly down-regulated β-catenin mRNA expression (<i>P</i> = 0.004). The expression of β-catenin mRNA expression in cells treated with 5-Aza-CdR reagent did not change significantly in the presence of high Kaiso expression (<i>P</i> = 0.062). Similar effects were observed in the LTE cell line (G and H; H: <i>P</i> = 0.003, <i>P</i> = 0.001, and <i>P</i> = 0.055 accordingly).</p

Figure 1. β-catenin mRNA expression was upregulated in lung cancer cell lines following treatment with 5-Aza-CdR.

<p>A: −1,124–11,114 bp revealed the presence of two CpG islands (positions −1,124–876 and 10,676–11,114). B: Treatment of lung cancer cell lines with 5-Aza-CdR resulted in varying levels of increased β-catenin mRNA expression. Statistical analysis by t-test showed increased β-catenin mRNA expression in lung cancer cell lines, which were treated with 5-Aza-CdR, compared to untreated cells (SPC, <i>P</i> = 0.030; H460, <i>P</i> = 0.308; LTE, <i>P = </i>0.035; A549, <i>P = </i>0.151).</p

Mutant 4 restored the E-cadherin to cell membrane and suppressed cell invasiveness in H460 cells.

<p>H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn, M1–M3, and M5-GFP was expressed in the nucleus and cytoplasm. GFP from GFP-si-P120ctn and M4-GFP was expressed in the nucleus, cytoplasm and on the cell membrane (IX). Expression or repletion of M4 mutant (IX) restored E-cadherin on the cell membrane (X), while expression of the other mutants had no significant effects on E-cadherin levels. (<b>B</b>) The levels of E-cadherin were then analyzed by Western blot assay. Expression of M1–M5 mutants were detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated the E-cadherin levels, whereas the other mutants had no effects on the E-cadherin levels. (<b>C</b>) The invasiveness of H460 cells was analyzed by Matrigel invasion assay. Repletion of M4 mutant reduced the cell invasiveness in comparison with the group of p120ctn ablation (Si-p120ctn) (**, <i>P</i><0.01), while repletion of M2 mutant enhanced the invasiveness (*, <i>P</i><0.01). Repletion of the other mutants (M1, M3 and M5) did not show significant effects on cell invasiveness in comparison with the group of 120ctn ablation.</p

Kaiso binds the β-catenin promoter region via methylated CpG dinucleotide sequences.

<p>No specific bands appear in the KBS binding region (A), while a specific band appears in the methylated CpG dinucleotide sequence region in SPC cell line (B). C: Luciferase reporter vectors and pRL-TK Vector were co-transfected into SPC cells with either the control vector or kaiso expressing plasmid DNA. They were then compared with cells treated with demethylating agents to assess the importance of the Kaiso binding domain. Statistical analysis by ANOVA indicated that the relative luciferase activity in the cell group with methylation site reporter vectors and Kaiso plasmid were higher than the other cell groups (<i>P</i> = 0.000, F = 83.018). No apparent changes in activity were observed in the SPC cells that were treated with demethylating agents (<i>P</i> = 0.374, F = 1.187). D: Luciferase activity obtained from the mutant construct showed no difference in any of these conditions (<i>P</i> = 0.674, F = 0.641). Similar results of ChIP (E, F) and luciferase analyses (G: <i>P</i> = 0.000, F = 37.703; <i>P</i> = 0.569, F = 0.718; H: <i>P</i> = 0.762, F = 0.513, respectively) were observed in the LTE cell line.</p