Figure 3
Effect of fibroblasts and conditioned media on E-cadherin and β-catenin (co)localization in PMC42-LA organoids. (a) Using immunocytochemistry and confocal microscopy, localization of E-cadherin was analyzed. Nuclei were visualized using ethidium bromide (red). (Part A) In control PMC42-LA organoids (no fibroblasts or conditioned media), E-cadherin staining was observed at cell junctions, which was confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture (right panel). (Part B) In NMF-conditioned medium E-cadherin label was detected as both junctional and cytoplasmic in PMC42-LA organoids, as confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture containing NMF-conditioned media (right panel). (Part C) In CAF-conditioned media E-cadherin was also detected as junctional and cytoplasmic in PMC42-LA organoids, with more predominant cytoplasmic localization. This was confirmed in 2-dimensional PMC42-LA culture containing CAF-conditioned media by confocal microscopy sectioning (right panel). With (part D) NMFs beneath filter or (part E) CAFs beneath the filter, E-cadherin was again detected at cell junctions and within cytoplasm. (b) E-cadherin and β-catenin are indicated by green and red label, respectively. Areas of colocalization appear yellow. (Part A) In control PMC42-LA cells (no fibroblasts or conditioned media), E-cadherin and β-catenin colocalized at cell junctions with some areas of non-colocalization. (Part B) When in NMF-conditioned medium, colocalization was detected at cell junctions with some independent localization. (Part C) In CAF-conditioned medium, E-cadherin localized to cytoplasm and β-catenin to cytoplasm and nuclei, with some overlap. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.