Fig. 6: NEK9-mediated selective autophagy of MYH9 is required for primary cilia formation.
a Schematic representation of the C-terminal regions of Homo sapiens NEK9 and deletion mutants. CC, coiled-coil. b Immunoprecipitation using MEFs stably expressing wild-type or deletion mutation NEK9 constructs after serum starvation (4 h). Data are representative of three independent experiments. c The C-terminal region of Homo sapiens NEK9. The putative MYH9-binding region is colored in magenta (top). Multiple sequence alignment of NEK9 from terrestrial vertebrates (bottom). See also Fig. 1c. d Immunoblotting of wild-type or Nek9-KO MEFs stably expressing indicated constructs. e, Quantification of the intensity of the MYH9 bands in (d). Data represent the mean ± SEM of three independent experiments. f Immunofluorescence microscopy of Nek9-KO MEFs stably expressing indicated constructs after serum starvation (24 h). g Frequency of ciliated cells in f. Data represent the mean ± SEM of five independent experiments (300 cells were counted in each experiment). h Quantification of cilia length in (f). Data were collected from 100 ciliated cells for each cell-type. i Immunofluorescence microscopy of Nek9W967A MEFs in which MYH9 was depleted by two independent shRNAs (#1 and #2). See Supplementary Fig. 6e, f for the knockdown efficiency of MYH9 in these cells. j Frequency of ciliated cells in (i), as in (g). Data represent the mean ± SEM of five independent experiments (300 cells were counted in each experiment). k Quantification of cilia length in (i), as in (h). Data were collected from 100 ciliated cells for each cell-type; p values correspond to a Tukey’s multiple comparisons test; *p < 0.0001. Scale bars, 10 µm and 3 µm (insets). Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile in (h, k).
