Fig. 4
From: CDC20B is required for deuterosome-mediated centriole production in multiciliated cells
CDC20B knockdown impairs multiciliogenesis in mouse ependymal MCCs. a, b Ependyma were stained for CDC20B (green) and FOXJ1 (nuclear MCC fate marker, red) 5 days post electroporation (5dpe) of control shRNA (a) or Cdc20b shRNA (b). sh277 is exemplified here, but all three Cdc20b shRNAs produced similar effects. c Graph showing the quantification of CDC20B protein levels in cells at the deuterosomal stage at 5dpe from two experiments. Mean values and standard error are shown. Unpaired t-test: ****p < 0.0001. d Dot plot showing the number of FOXJ1-positive nuclei observed for each field, with mean values and standard deviations from two experiments. Unpaired t-test: p = 0.3961 (sh273, ns), p = 0.1265 (sh274, ns), p = 0.3250 (sh277, ns). No significant variations were observed between conditions, indicating that MCC fate acquisition was not affected by Cdc20b knockdown. e, f Confocal pictures of 9dpe ependyma electroporated with control shRNA (e) or Cdc20b shRNAs (f) and stained for DEUP1 (deuterosome, green), FOP (centrioles, red) and ZO1 (cell junction, white). DEUP1-positive deuterosomes with non-disengaged FOP-positive centrioles were observed much more frequently in MCCs electroporated with Cdc20b shRNAs compared to control. g Dot plot showing the percentage of MCCs with non-disengaged centrioles per field, with mean values and standard deviations. Two experiments were analyzed. Unpaired t-test: ****p < 0.0001. h, i Confocal pictures of 15dpe ependyma stained for FOP (centrioles, green), α-Tubulin (α-TUB, cilia, red), and ZO1 (cell junction, white) showing the morphology of normal MCCs in shRNA control condition (h), and examples of defects observed in MCCs treated with sh Cdc20b (i). j Dot plot showing the number of released centrioles per cell, with mean values and standard deviations. k Dot plot showing the percentage of normal and abnormal MCCs per field of observation, with mean values and standard deviations. MCCs were scored abnormal when they did not display organized centriole patches associated to cilia. Three experiments were analyzed. Unpaired t-test: p = 0.0004 (sh273, ***), p = 0.0001 (sh274, ****), p = 0.0038 (sh277, **). Scale bars: 20 μm (a), 5μm (e, i)
