Skip to main content
. 2017 Sep 12;4:149. doi: 10.3389/fvets.2017.00149

Figure 1.

Figure 1

Morphology and expression of lineage-specific markers in canine mammary tumor (CMT)-U27 and -U309 cell lines. (A) CMT-U27 and -U309 cells exhibit distinct morphological features, U27 cells had more variation in cell size and shape, while U309 were mostly elongated and typically not contact inhibited by the confluency. Actin cytoskeleton is stained with phalloidin and nuclei with DAPI. Scale bar 10 μm. (B) Western Blotting from cellular lysates revealed that epithelial cell–cell contact proteins E-cadherin and claudin-1 are absent from CMT-U309 cells, while these cell express N-cadherin. In contrast, CMT-U27 cells possess expression of both E-cadherin and claudin-1, typical for luminal epithelial cells. Western Blot experiments were repeated at least three times. (C) Proliferation rates and average cell doubling times of CMT-U27 and CMT-U309 cell lines were measured from sparsely growing cultures in an o/n experiment (see Materials and Methods for details). Values are presented in box-plots, where the median is indicated by the central bar. 5/95 percentile whiskers with outliers are shown; n = 3–6. (D) Western blot experiments from the cellular lysates of CMT-U27 and -U309 cell lines showed that CMT-U309 cells express both p63 and slug, typical markers for the basal breast epithelial cells, while CMT-U27 cells possess almost non-detectable levels of these proteins. Western Blot experiments were repeated at least three times. (E) Mesenchymal marker vimentin is highly expressed in CMT-U309 cells as shown by western blots from the cellular lysates of CMT-U27 and -U309 cells as well as immunofluorescence stainings with anti-vimentin antibody (F). Nuclei are visualized with DAPI. Scale bar 10 μm. (G) Expression of cytokeratins was studied with anti-CK5 anti-CK14 and anti-Pan-Keratin antibody recognizing CK4, 5, 6, 8, 10, 13, 18 by Western Blotting from cellular lysates. Expression of CK5 was detected in both studied cell lines, while CK14 was detected only in CMT-U309 cells. Detection with Pan-Keratin ab showed differences in the expression of CK8 and 18 (53 and 48 kDa, respectively) that are typical for the luminal epithelial cells. Western Blot experiments were repeated at least three times.