Figure 4: Functional relevance of HOXC4 and AID in the fallopian tube.

(a) FFPE sections of proximal and fimbrial compartments of fallopian tubes from BRCA carriers (n=13) and non-carriers (n=14) were stained with an anti-HOXC4 antibody and scored for signal intensity. P-values were calculated with the Wilcox-test; the box shows the upper and lower quartiles and the whiskers show 1.5 X inter-quartile range beyond the upper and lower quartiles. (b, left) Primary cultures of secretory epithelial cells isolated from the fimbrial compartment of the fallopian tube (FT3, FT4, FT8 and FT11) and long-term cultures of SKOV3 ovarian carcinoma cells were treated with TNF-α or IL1-β for 6 h. Relative expression of AID mRNA was analysed by real-time PCR; data from three replicates are shown. (right) Relative expression of AID mRNA determined by real-time PCR in SKOV3 cells stably expressing HOXC4 shRNA or transfected with an empty vector and treated with TNF-α or IL1-β for 6 h; data from three replicates are shown. P-values were calculated with the t-test; the error bars indicate one standard deviation. (c) SKOV3 cells stably overexpressing HOXC4 or empty vector were treated with Hoechst 3342 (5 μg ml⁻¹) for 90 min and Hoechst low-staining cells (side population) were examined by flow cytometry; data from three replicates are shown. P-values were calculated with the t-test; the error bars indicate 1 s.d. (d) HOXC4 mRNA expression determined by real-time PCR in SKOV3 cells stably overexpressing AID or transfected with an empty vector; data from three replicates are shown. P-values were calculated with the t-test; the error bars indicate 1 s.d. (e) AID protein expression in FFPE sections of proximal and fimbrial compartments of the fallopian tube of BRCA carriers (n=13) and controls (n=14) analysed by immunohistochemistry and scored for signal intensity. P-values were calculated with the Wilcox-test; the boxes show the upper and lower quartiles and the whiskers show 1.5 X inter-quartile range beyond the upper and lower quartiles. (f) Representative AID immunostains (× 200 magnification). Bars indicate 200 μm. (g) Primary cultures of secretory epithelial cells from the fimbrial fallopian tube compartment (FT7, FT8 and FT11), and of immortal cultures of HOC7 and SKOV3 ovarian carcinoma cells stably overexpressing AID or transfected with an empty vector were treated with Hoechst 3342 (5 μg ml⁻¹) for 90 min and analysed by flow cytometry to evaluate the Hoechst low-staining cells (side population); data from three replicates are shown. P-values were calculated with the t-test; the error bars indicate 1 s.d.