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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Dev Biol. 2015 Oct 23;409(1):202–217. doi: 10.1016/j.ydbio.2015.10.023

Figure 5. Luminal PTEN loss results in misoriented mitotic spindles, without altering cell-cell adhesion or apical polarity.

Figure 5

(A–C) IF staining was performed to detect cells in anaphase (pH3) or telophase (Ki67) combined with K8 or K14 to distinguish luminal and basal cells, respectively. (A) Mitotic spindle angles were measured by drawing a line through the centers of the condensed chromatin of the daughter nuclei of cells in anaphase or telophase and defining it as the spindle axis. The spindle angle was measured at the intersection between the spindle axis and a line drawn parallel to the basement membrane. (B) Quantification of the spindle angles demonstrated that luminal PTEN loss did not alter the mitotic spindle orientation of dividing basal epithelial cells, and the majority of the divisions occurred parallel to the basement membrane (spindle angle < 15º). In the luminal epithelium, the K8PTEN-KO ducts showed a 4-fold increase in mitotic spindle angles compared with the K8-CTR ducts. The n indicates individual mitotic events. (C) The quantification of mitotic spindle angles was further analyzed to determine differences between single- and multi-layered luminal epithelium in the K8PTEN-KO ducts. Representative low and high magnification images show mitotic Ki67-positive luminal cells from K8-CTR (left image with green inset), single-layered K8PTEN-KO (center image with pink inset), and multi-layered K8PTEN-KO (right image with blue inset) ducts. Scale bars: 50 μm (low magnification images) and 10 μm (high magnification images). Below each set of images is their corresponding spindle angles plotted as percentages in 15º increments. In the K8-CTR ducts, 81.3% of the luminal cells showed parallel divisions, whereas in the single- and multi-layered K8PTEN-KO ducts only 47.1% and 12.7%, respectively, showed parallel divisions. The n indicates mitotic events, and the mean spindle angles are shown below each angle plot. (D) Representative confocal IF images depict similar staining patterns of the cell-cell adhesion marker E-cad, and the apical polarity marker aPKC, between K8-CTR and K8PTEN-KO ducts. Scale bars: 50 μm. All K8-CTR images shown in this figure are from K8-CTR-Veh mice.