Fig. 6

ANLN is essential for mitotic rounding early in the developing epidermis. A Upper panel: whole-mount immunofluorescence images of control (Ctrl) and shAnln-926-transduced E14.5 embryos immunolabeled for E-cadherin and imaged at the middle of the basal layer. Lower panel: Computer segmentation of E-cadherin staining to visualize cell borders. B Quantification of cell areas from the data shown in A. n = 2746 and 2759 Ctrl and shAnln-926-transduced cells, respectively, from three embryos per condition. Horizontal bars represent the mean, and circles represent individual cells. NS denotes not significant. C Quantification of axial ratio from the data shown in A. n = 539 and 560 Ctrl and shAnln-926-transduced cells, respectively, from three embryos per condition. Horizontal bars represent the mean, and circles represent individual cells. NS denotes not significant. D Whole-mount immunofluorescence of control and Anln-926 KD E14.5 embryos immunostained for the adhesion receptor E-cadherin and imaged at the middle of the basal layer. Arrows denote early mitotic cells. E Quantification of the early mitotic cell axial ratio from the data shown in D. n = 77, 95, and 99 Ctrl, Anln-926- and Anln-2981-transduced cells, respectively, from four embryos per condition. Horizontal bars represent the mean, circles represent individual cells. P < 0.0001 for Ctrl versus Anln-926; P < 0.0001 for Ctrl versus Anln-2981 by unpaired two-tailed t-test. F Whole-mount immunofluorescence images of Anln-GFP-transduced E16.5 embryos immunolabeled for E-cadherin and imaged at the middle of the basal layer. Arrows denote early mitotic cells. G Whole-mount samples were treated as in A. H2B-GFP (green) indicates infected cells. Arrows denote cells with abnormal chromosome segregation. H Quantification of the mitotic stage from the data shown in G. Horizontal bars represent the mean, and circles represent individual microscopic fields. n = 42 and 39 Ctrl and Anln-926 microscopic fields, respectively, from four embryos per condition. P = 0.003 by unpaired two-tailed t-test. I Quantification abnormal chromosome segregation from the data shown in G. n = 3 embryos per condition. P = 0.0319 by unpaired two-tailed t-test. Horizontal bars represent the mean ± SD, and circles represent the mean of cells with abnormal chromosome segregation in 0.5cm² dorsal skin E14.5 embryos. In A and D, insets show the transduced cells (H2B−GFP+). Nuclei were stained with DAPI (blue). Scale bars = 20 μm