Differences between <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN group compared to <i>Matn2^-/-</i> control.

<p>Differences between <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN group compared to <i>Matn2^-/-</i> control.</p

Results of Phospho-RTK antibody array.

<p>Picture of RTK array membrane (<b>A</b>). Densitometry of phosphorylation signals in WT control (dark gray bars) and WT DEN-treated (white bars) samples, compared to control (black bars) and <i>Matn2^-/-</i> DEN-treated (light gray bars) samples (<b>B</b>). Data are expressed as mean ± SD, n = 3.</p

Results of DEN treatment in WT and <i>Matn2^-/-</i> animals.

<p>Representative pictures of the macroscopic appearance of WT and <i>Matn2^-/-</i> DEN-treated mouse livers. 10 months after DEN exposure the number and size of macroscopic tumors were greater in <i>Matn2^-/-</i> than in WT livers (<b>A</b>). The number of tumors/animal (<b>B</b>) and the tumor volume (<b>C</b>) were significantly higher in <i>Matn2^-/-</i> mice compared to WT mice after DEN-treatment (n = 9 for WT DEN, n = 13 for <i>Matn2^-/-</i> DEN, **p<0.01).</p

Immunolocalization of Matn2 in the liver of young mice (A).

<p>Matn2 immunostaining is most intense around the portal blood vessels (arrowhead) on a frozen section of a 40-day old WT mouse liver. Matn2 partially colocalizes with laminin; however, in several structures Matn2 staining is more intense (arrowheads). There is no immunosignal in the matching area in the liver of a <i>Matn2^-/-</i> mice. Bar, 0.1 mm. <b>Representative histological stain of WT control, WT DEN-treated, </b><b><i>Matn2^-/-</i></b><b> control and </b><b><i>Matn2^-/-</i></b><b> DEN-treated mouse livers (B).</b> Scale bars represent 0.1 mm.</p

Representative Western blots of intracellular regulatory proteins

<p>in WT control, WT DEN-treated, <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN-treated mouse livers (<b>A/1, A/2</b>). Results of densitometrical analysis of band intensities expressed as values normalized to β-Actin loading control (<b>B</b>). Data are expressed as mean ± SD, n = 3.</p

Representative immunohistochemical stains of WT control, WT DEN-treated, <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN-treated mouse livers (A).

<p>Scale bars represent 0.1(insets) for paraffin-embedded samples. Changes in cell cycle regulation. Ki-67 proliferation index (B). In knockout samples an average of 4.1 Ki-67 positive cells were counted per field of view compared to 0.7 in WT (p<0.001) (B). Results are expressed as mean ± SD. <b>Representative Western blots of cell cycle regulatory proteins (C) in WT control, WT DEN-treated, </b><b><i>Matn2^-/-</i></b><b> control and </b><b><i>Matn2^-/-</i></b><b> DEN-treated mouse livers.</b> Diagrams of band intensities expressed as values normalized to β-Actin loading control (D). Data are expressed as mean ± SD, n = 3.</p

Differences between WT control and WT DEN group compared to WT control.

<p>Differences between WT control and WT DEN group compared to WT control.</p

Differences between WT control and <i>Matn2^-/-</i> control group compared to WT control.

<p>Differences between WT control and <i>Matn2^-/-</i> control group compared to WT control.</p

Representative immunofluorescence and immunohistochemical stains of WT control, WT DEN-treated, <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN-treated mouse livers.

<p>Scale bars represent 0.1(insets) for paraffin-embedded samples and 0.05 mm for frozen tissue sections.</p

The expression of <i>GCG</i>, <i>NMES-1</i>, <i>LRMP</i>, and <i>FAM161B</i> genes, tested on independent laser microdissected colonic and also on demethylated HT-29 cells by real-time PCR.

<p>Data analysis was carried out with the comparative Cp method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046215#s4" target="_blank">Materials and Methods</a>). Genes were considered to be downregulated with values lower than 0.5 (50% decrease, horizontal dotted line), and upregulated with values higher than 2 (two-fold increase). GAPDH was used as a control housekeeping gene. Light grey and dark grey columns show gene expression levels in adenoma and in tumor samples relative to normal samples, respectively. Hatched columns indicate the comparison of 5-Aza treated and control cells. Standard deviations of the measured transcript levels were calculated and are indicated for each transcript. Housekeeping gene intensities were averaged for each group.</p