Fig. 8
Embryonic signals affect vesicular trafficking within luminal epithelial cells. A The epithelial origin of primary cell culture was tested using a cytokeratin marker. Actin filaments were marked with phalloidin. The nucleus was stained with DAPI. B Identification of regions of interest (ROIs) was performed based on the colocalization of CD63 (CY3, red) and VPS36 (Alexa 488, green) at the cell-to-cell contact areas. The nucleus was stained with DAPI. Merged images show an example of ROIs (white arrows). C A representative immunofluorescence image displaying luminal epithelial cell colony evaluated for intestines and colocalization. D Signal intensities for CD63 and VPS36 after treatment with embryonic signals—E2 or PGE2 (100 nM each; unpaired T-test, p < 0.05). E A heatmap of positively colocalized pixels of both proteins (Cy3 for CD63 and Alexa 488 for VPS36). Values are reported as pairs of pixels that range from 0 to 1 (no to full colocalization). F Scatter plots of CD63 + and VPS36 + pixels corresponding to the colocalization events as shown in B and C indicate correlation and linear relationship for control (r = 0.87, p = 0.0002) and lack of correlation and non-linear relationships for E2 (r = 0.11, p = 0.613), PGE2 (r = 0.39, p = 0.073) and a simultaneous treatment with E2 and PGE2 (r = 0.32, p = 0.114). A Pearson correlation coefficient was performed (p < 0.05)