RUNX3, CD8 and ThPOK triple fluorescence.

<p>Triple colocalization of RUNX3, CD8 and ThPOK in NM (panel A-D, Scale bar = 50 µm), MA (panel E-H, Scale bar = 30 µm) and CRC (panel I-L, Scale bar = 30 µm). RUNX3: green (panel A, E, I); CD8: red (panel B, F, J); ThPOK: blue (panel C, G, K). Merge (panel D, H, L): CD8+ cells expressing RUNX3: yellow (arrow in panel H); CD8+ cells coexpressing RUNX3 and ThPOK: white (arrows in panel L).</p

Immunofluorescence quantification by confocal analysis.

<p>Fluorescence quantification (ImmunoFluorescence Intensity Score, IFIS, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054488#s2" target="_blank">Materials and Methods</a>) of CD4, CD8, CD56 and ThPOK in normal colorectal mucosa (NM), microadenoma (MA) and colorectal carcinoma (CRC). * p<0.05 vs normal colorectal mucosa.</p

Foxp3, GZMB and RUNX3 fluorescence levels.

<p>Fluorescence levels of Foxp3 (panel A), GZMB (panel B), RUNX3 (panel C) immunostaining. *P<0,05 vs NM; ‡ P<0,05 vs CRC. RUNX3 level in CD8+ T cells coexpressing (white bars) and not coexpressing (black bars) ThPOK in NM, MA and CRC (panel D). *P<0,05.</p

Confocal immunofluorescence staining.

<p>Examples of confocal analysis of cryosections of normal colorectal mucosa (NM), microadenoma (MA), and colorectal carcinoma (CRC), labelled by DAPI (blue), ThPOK (red), CD4 (green), CD8 (green), and CD56 (green). Double immunolabelled cells appear as yellow spots. <b>Panels A-C</b>: Colocalization imaging of ThPOK with CD4 in NM (panel A), MA (panel B) and CRC (panel C). <b>Panels D-F</b>: Double immunolabelling performed by ThPOK and CD8 in NM (panel D), MA (panel E) and CRC (panel F). <b>Panels G-I</b>: Immunostaining with ThPOK and CD56 in NM (panel G), MA (panel H) and CRC (panel I). Scale bar  = 80 µm.</p

ThPOK protein and mRNA during colorectal neoplastic progression.

<p>Measurement of ThPOK protein and mRNA in normal colorectal mucosa (NM), microadenomas (MA), and colorectal cancer (CRC). <b>Panel A:</b> NM, MA and CRC were subjected to SDS–PAGE/western blot using the anti-ThPOK antibody; densitometric analysis and bands at 60 kD for ThPOK and at 42 kD for α-actin.* P<0.05 vs NM. <b>Panel B:</b> Real-time PCR analysis of ThPOK mRNA in NM, MA and CRC. *P<0,05 vs NM. Band intensity of ThPOK protein and ThPOK mRNA level for NM were arbitrarily set to 1.</p

Colocalization analysis.

<p>Quantitative analysis of co-expression levels by Manders coefficient in normal mucosa (NM), microadenomas (MA), and colorectal cancer (CRC), analyzing the ratio between ThPOK/CD4 (panel A), ThPOK/CD8 (panel B), and ThPOK/CD56 (panelC). *P<0,05 vs NM; ‡ P<0,05 vs CRC. Panel D: Normalized co-expression levels in normal mucosa (NM), microadenomas (MA), and colorectal cancer (CRC), between ThPOK and CD4 (white bars), ThPOK and CD8 (black bars), and ThPOK and CD56 (gray bars). *P<0,05 vs CD4; ‡ P<0,05 vs CD56.</p

Additional file 2: Table S2. of Aberrant DNA methylation profiles of inherited and sporadic colorectal cancer

Details of FISH probes used for CIN analysis. (DOCX 13.5 kb