Fig. 7

ANLN is essential for mitotic rounding in cultured keratinocytes. A Wild-type keratinocytes were transduced with shScr (Ctrl) or shAnln-926, treated with nocodazole for 6 hours, fixed, and labeled for F-actin. B Quantification of the early mitotic cell axial ratio from the data shown in A. Horizontal bars represent the mean, and circles represent individual cells. n = 181, 176, and 155 Ctrl, Anln-926-, and Anln-2981-transduced cells, respectively, from four experiments per condition. P < 0.0001 for Ctrl versus Anln-926, P < 0.0001 for Ctrl versus Anln-2981 by unpaired two-tailed t-test. C Wild-type keratinocytes immunolabeled for ANLN. D Wild-type keratinocytes treated as in A and immunostained for F-actin. Quantification of the normalized cortical intensity is presented to the right of the image. n = 76 and 76 Ctrl and Anln-transduced cells, respectively, from three experiments. P = 0.0019 for Ctrl versus Anln-926 by unpaired two-tailed t-test. E Wild-type keratinocytes treated as in A and immunostained for myosin IIa. Quantification of the normalized cortical intensity is presented to the right of the image. n = 62 and 67 Ctrl and Anln-transduced cells, respectively, from three experiments. P = 0.0148 by unpaired two-tailed t-test. F Wild-type keratinocytes treated as in A and immunostained for pMLC. Quantification of the normalized cortical intensity is presented to the right of the image. n = 70 and 68 Ctrl and Anln-transduced cells, respectively, from three experiments. P < 0.0001 for Ctrl versus Anln-926 by unpaired two-tailed t-test. G Wild-type keratinocytes treated as in A and immunostained for pERM. Quantification of the normalized cortical intensity is presented to the right of the image. n = 64 and 68 Ctrl and Anln-transduced cells, respectively, from three experiments. P = 0.0061 for Ctrl versus Anln-926 by unpaired two-tailed t-test. D–G Horizontal bars represent the mean, and circles represent individual cells. H Representative images of GFP-rGBD in shScr (ctrl) or Anln-926-transduced primary mouse keratinocytes. Arrows indicate the changes of active RhoA localization. Nuclei were labeled with Hoechst. The dotted line denotes cell edge. Scale bars = 10 μm. I Line scan analyses from data shown in H, t = 4 min. J Quantification of cortical coverage of GFP-rGBD in early mitotic cells. Horizontal bars represent the mean. n = 13 and 14 Ctrl and Anln-KD 1⁰MK, respectively, from three independent experiments. P = 0.0005 by unpaired two-tailed t-test. K Wild-type keratinocytes were co-treated with nocodazole and DMSO (Ctrl) or nocodazole and jasplakinolide, or nocodazole and calyculin A, fixed, and labeled for F-actin and pERM. L Quantification of mitotic cell axial ratio from the data shown in K. Horizontal bars represent the mean, and circles represent individual cell. n = 86, 93, and 93 DMSO, jasplakinolide, and calyculin A-treated cells, respectively, from three experiments. P<0.0001 for DMSO versus jasplakinolide; P < 0.0001 for DMSO versus Calyculin A by unpaired two-tailed t-test. M Whole-mount immunofluorescence images of wild-type E15.5 embryos treated with DMSO (Ctrl), jasplakinolide, or Calyculin A, fixed, and immunolabeled for E-cadherin and imaged at the middle of the basal layer. The dotted line denotes cell edge. N Quantification of early mitotic cell axial ratio from data shown in M. n = 39, 37, and 43 DMSO, jasplakinolide, and Calyculin A, respectively, from three treated embryos, per condition. Horizontal bars represent the mean, and circles represent individual cells. P < 0.0001 for DMSO versus jasplakinolide; P = 0.0034 for DMSO versus Calyculin A by unpaired two-tailed t-test. In H, K, and M, insets show the nuclei in a grayscale. Nuclei were stained with DAPI (blue). Scale bars = 20 μm