Prognostic value of connexin protein isotype expression using multivariate Cox regression analysis.

<p>HR: hazard ratio; CI: confidence interval;</p><p>0123: score categories where _ represents thresholds.</p><p>Prognostic value of connexin protein isotype expression using multivariate Cox regression analysis.</p

Kaplan-Meier plots of significant correlations between overall survival (OS) and elevated (>median) Cx30 (a–c), Cx43 (d–f) and Cx46 (g–i) mRNA expression in 1988 breast cancers based on <i>in silico</i> analysis of Illumina array data.

<p>Elevated Cx30 levels were linked with reduced OS in patients with luminal B tumors (a) and in ER positive endocrine therapy treated patients (b), but with a strong tendency for improved OS in ER negative cases (c). Cx43 expression was associated with better OS in the whole cohort (d) and in ER positive endocrine treated patients (e) but showed a strong trend for reduced OS in ER negative patients (f). Cx46 expression was associated with improved OS in the whole cohort (g), in ER negative patients (h) and in HER2 positive patients (i). Significance (at p<0.05) was calculated using the log-rank test. HR: hazard ratio (at 95% Confidence Interval).</p

Kaplan-Meier plots of significant prognostic correlations of elevated (>median) Cx43 (a-d and g–i) and Cx46 (e–f) mRNA expression in 1809 breast cancers based on <i>in silico</i> analysis of Affymetrix array data.

<p>Cx43 expression was associated with improved relapse/disease free survival (RFS) in a tumor group similar to SEER prevalence (a), in ER and lymph node positive tumors (b) and in ER positive endocrine treated tumors (c), but reduced RFS in ER negative tumors (d). Elevated Cx46 levels were predictive for better RFS in the whole patient cohort (e) and in ER and lymph node positive grade 3 patients (f). Elevated Cx43 mRNA levels were also correlated with significantly better distant metastasis-free survival (DMFS) in ER positive endocrine treated patients (g) and in grade 2 cancers (h). Cx43 expression in ER negative patients was linked with reduced overall survival (OS) (i). Significance (at p<0.05) was calculated using the log-rank test. HR: hazard ratio (at 95% Confidence Interval).</p

Detection of connexin protein isotypes (Alexa-564, red) and the proliferation marker Ki67 protein (Alexa-518, green) in normal pre-menopausal breast tissue.

<p>Punctuate Cx43 reaction was localized to the myoepithelial cell layer of normal mammary glands and to adjacent stromal cells (arrow) (a). Both Cx32 (b and c) and Cx26 (d) proteins were found dominantly in the luminal epithelial cells. Cx46 protein was found both in the myoepithelial and luminal cells and less in stromal inflammatory cells (arrow) (e and f). High power views show Cx32 mainly linked to the luminal cells (c) and Cx46 mainly localized to the basal cells (f) involving both their cytoplasm and intercellular borders. Cx30 was revealed along myoepithelial cells and at the apex of luminal epithelium and less in the rest of luminal cells, and along vascular endothelial cells (arrow) (g). Co-localization of Cx26 (red) and Cx30 (green) in epithelial cells (yellow), involving the intercellular borders (h). Double immunofluorescence, cell nuclei are stained blue using Hoescht. Summary drawing of our results shows potential homo- or heterocellular/typic interactions of Cx26 (red), Cx32 (blue), Cx46 (violet), Cx43 (green) and Cx30 (yellow) gap junctions in a normal mammary gland (i).</p

Examples of numerical chromosomal and telomeric alterations in GCTB stromal cells of male patients.

<p>Centromeric 3 (red), 4 (green), 6 (yellow) and X (light blue) signals show different levels of polysomy. Chromosome 4 trisomy in a cell disomic for the rest (3,6 and X) of the tested centrosomes (b) and chromosome 11 subtelomeric loss and tetrasomy in a cell of another case (c). Scale bar on <u>a</u> represents 5 μm; and 2.5 μm on <u>b</u> and <u>c</u>.</p

Immunoperoxidase (a-c) and immunofluorescence (d-e) detection in osteoclast rich areas and surrounding stroma (f and g), and clinicopathological correlations of Cx43 protein levels (h and i) in giant cell tumor of bone.

<p>Examples of tumors with moderate (a; score 3) and high (b; score 8) Cx43 levels in mononuclear cells. Strong Cx43 reaction in the preexisting osteoblast layer around bone spicules and in osteocytes (arrowhead) (c). A tumor nest and adjacent ring of reactive stroma are annotated separately for counting Cx43 (Alexa564, red) plaques (d; OC-osteoclasts). Higher power of (d) with osteoclasts encircled (e). Digital image segmentation highlights Cx43 plaques in orange for automated counting (f). Both the Cx43 positive area fraction (g) and the number of Cx43 positive plaques (h) are significantly reduced within tumor nests (p<0.01). Cx43 levels are also significantly reduced in aggressive vs active and in aggressive vs latent clinicoradiological tumor stages (i). Scale bar on (a) represents 30 μm on <u>a</u>, <u>b</u> and <u>c</u>; 80 μm on <u>d</u>, 30 μm on <u>e</u> and 15 μm on <u>f</u>.</p

Kaplan-Meier plots of univariate Cox regression analysis of Cx43 immunoscores in giant cell tumor of bone.

<p>An increased hazard of progression (reduced PFS) is linked to scores 1–3 vs 4–8 (arrow) separating patient number around the median, N_score1-3 = 60 (48.8%), N_score4-8 = 63 (51.2%) (a). Log-rank test proves significantly reduced progression free survival (PFS) in tumors presenting low (scores 1–3) vs high (scores 4–8) Cx43 protein levels (b).</p

Dye coupling test for measuring potential communication through gap junctions with flow cytometry.

<p>Scheme on the principle of the technique (a). Unlabelled cell are mixed with double dye labelled cells (orange) of the same kind at a ratio of 10:1 (a-b). Calcein (Mw:622 Da, green), after esterase cleavage becomes hydrophylic and can pass into adjacent cells through gap junctions, while the larger lypophylic DiI (red) is trapped within donor cell membranes (b). The proportion of single calcein labelled cells measured with flow cytometry (B+-, lower right box) indicating dye coupling, is significantly higher (p<0.001) in the control cell cultures (c) than in GCTB stromal cell cultures (d). Diagram showing the mean ± standard deviation of dye transfer in 3 independent experiments using stromal cells isolated from 3 patients (e). Scale bar on <u>b</u> represents 20 μm.</p

Detection of Cx43 levels and the subcellular distribution of Cx43 protein in primary GCTB stromal cell, bone marrow stromal (BM) cell and HDFa fibroblast cultures.

<p>Immunoperoxidase reaction reveals paranuclear clumps of Cx43 protein in the frequently binucleated neoplastic GCTB stromal cells (arrows) (a). Significantly less Cx43 is linked to cell membranes in GCTB stromal cells than in the control cells as tested using immunofluorescence (b-g; red) and digital image analysis (b). Arrowheads highlight characteristic localization of Cx43 in the endoplasmic reticulum-Golgi region in GCTB stromal cells (b and c, identical areas) and in cell membranes in HDFa fibroblasts (d and e, identical areas). Cx43 is dispersed throughout bone marrow stromal cells including cell membranes (f and g, identical areas). Vimentin reaction in b, d and f (green) highlights cell shape, while black and white images of identical areas (c, e and g) better reveal subcellular localization of Cx43. Cx43 transcript and protein levels detected using RT-PCR (i) and western blots (j), respectively. In western blots, control cells but not GCTB stromal cells show alkaline phosphatase sensitive bands (P1 and P2). Results in graphs show the mean ± standard deviation of three independent experiments. For blue nuclear staining hematoxylin (a) and Hoescht (b and d and f) were used. Scale bar on <u>a</u> represents 20 μm; and 10 μm on b, <u>c</u>, <u>d</u>, <u>e</u>, and 15 μm on <u>f</u> and <u>g</u>.</p

Significant prognostic correlations of connexin mRNA expression data resulting from the <i>in silico</i> analysis of 1809 (Affymetrix) and 1988 (Illumina) breast cancers.

<p>A: Affymetrix platform using relapse-free survival (RFS) data if not indicated, distant metastasis-free survival (DMFS) data indicated as M or overall survival data (OS).</p><p>I: Illumina platform using OS data only (not indicated).</p><p>Significant prognostic correlations of connexin mRNA expression data resulting from the <i>in silico</i> analysis of 1809 (Affymetrix) and 1988 (Illumina) breast cancers.</p