Nuclear co-localization of CDX2 and SPANX-B in differentiating Caco-2 cells.

<p>Immunofluorescent staining of differentiating Caco-2 cells with DAPI counterstaining reveals overlapping SPANX-B (Alexa Fluor 488: green) and CDX2 (Alexa Fluor 568: red) expression; (20× magnification) (<b>A</b>). More than 60 to 80% of the cells show double-labeling when analyzed quantitatively (<b>B</b>).</p

Up-regulation of CT genes in parallel to MET in the Caco-2 SD model.

<p>Relative mRNA expression of CT genes (<i>PAGE2</i>, <i>-2B</i> and <i>SPANXB</i>) (<b>A</b>), epithelial genes (<i>E-cadherin (CDH1)</i>, <i>claudin 4(CLDN4)</i>, <i>CDX2</i>) (<b>B</b>), and mesenchymal genes (<i>fibronectin 1 (FN1), vimentin (VIM)</i>, <i>transgelin (TAGLN)</i>) (<b>C</b>) as determined by quantitative PCR at days 0, 10, 20 and 30 post-confluence. Data represent average of two experiments. Change in expression levels for all genes between days 0 and 30 is statistically significant (P<0.0001, by two way ANOVA with Tukey's post hoc test).</p

<i>TET</i> expression during Caco-2 SD.

<p>mRNA of all three <i>TET</i> genes increase gradually during Caco-2 SD (<b>A</b>). An increase in only a 25 kD version (<b>B</b>), but not the full-length TET2 protein (<b>C</b>) occurs simultaneously with the increase in mRNA. BAPTA-AM treatment results in a modest decrease in the 25 kD TET2 protein, with the generation of a larger mw version (<b>D</b>). P values, as determined by one-way ANOVA, are 0.03, 0.01, and 0.07, for Tet1, Tet2, and Tet3, respectively.</p

Increased hydroxymethylation of <i>PAGE2</i> and <i>SPANX-B</i> during Caco-2 spontaneous differentiation.

<p>CHIP experiments using an anti-hmC antibody and primers corresponding to +31 to +182 and +68 to +184 bp from the transcription start site of the <i>PAGE2</i> and <i>SPANX-B</i> genes, respectively. P values (one-way ANOVA) are 0.001 and 0.07 for PAGE2 and SPANX-B, respectively.</p

SPANX-B and vimentin expression are mutually exclusive in differentiating Caco-2 cells.

<p>Immunofluorescent staining of differentiating Caco-2 cells with DAPI counterstaining reveals a gradual increase in nuclear SPANX-B (green) with a concomitant decrease in cytoplasmic vimentin expression (red); (20× magnification) (<b>A</b>). Less than 1% of SPANX-B positive cells showed staining for vimentin on day 0 (<b>B</b>).</p

Bisulfide sequencing of <i>PAGE2, -2B</i> and <i>SPANX-B</i> promoter-proximal regions.

<p>Filled and empty circles represent methylated and unmethylated cytosines, respectively. % methylation within each analysed region, based on the 10 clones sequenced is indicated. CpG residues proximal to <i>PAGE2, -2B</i> and <i>SPANX-B</i> promoters during Caco2 differentiation at days 0, 10 and 30 reveals no statistically significant change (by one-way ANOVA).</p

Overlapping TET2 and CT gene expression in differentiating Caco-2 cells.

<p>Double immunofluorescence staining for TET2 (Alexafluor 568: red) and SPANX-B or PAGE2 (Alexafluor 488: green) with DAPI counterstaining 20 days post-confluence show overlapping nuclear expression Magnification: 40× (<b>A</b>). More than 95% of cells expressing PAGE2 or SPANX-B were also positive for TET2 staining (<b>B</b>).</p

De-differentiation induced EMT and down-regulation of <i>CT</i> and <i>TET</i> genes.

<p>De-differentiation induced by growth under non-confluent conditions indicated by decreased <i>sucrose isomaltase</i> (SI) mRNA levels (<b>A</b>), leads to down-regulation of <i>CDX2</i>, <i>PAGE2, -2B</i> and <i>SPANX-B</i>, with concomitant up-regulation of <i>TGLN</i> (<b>B</b>). <i>TET1</i> and <i>-2</i> mRNAs are also down-regulated during de-differentiation (<b>C</b>).</p