Fig. 8: Autophagic degradation of NEK9–MYH9 and OFD1 is required for primary cilia formation.
a FRAP analysis of GFP-actin in wild-type MEFs stably expressing FLAG or FLAG-OFD1 after serum starvation (24 h). Images were recorded at 5-s intervals following photobleaching of the indicated area, and fluorescence recovery at different time points was quantified. Data represent the mean ± SEM of 10 cells. b Immunofluorescence microscopy of wild-type or Nek9W967A MEFs after serum starvation (24 h). OFD1 or MYH9 was depleted by shRNA-mediated knockdown (shMYH9 #1 was used). Similar results were obtained using shMYH9 #2 and two independent shOFD1 (not shown). See Supplementary Fig. 8b–d for the knockdown efficiency. c Percentage of cells with centriolar satellites OFD1 in (b). Data represent the mean ± SEM of three independent experiments (100 cells were counted in each experiment). d Frequency of ciliated cells in (b). Data represent the mean ± SEM of five independent experiments (300 cells were counted in each experiment). e Quantification of cilia length in (b). Data were collected from 100 ciliated cells for each cell-type. Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile. f Immunofluorescence microscopy of wild-type or Fip200-KO MEFs after serum starvation (24 h). OFD1 and/or MYH9 were depleted by shRNA-mediated knockdown. See Supplementary Fig. 8e–g for the knockdown efficiency. g Frequency of ciliated cells in (f), as in (d). h Quantification of cilia length in (f), as in (e); p values correspond to a Tukey’s multiple comparisons test; *p < 0.0001. Scale bars, 10 µm and 3 µm (insets). i Model of how autophagy drives primary cilia formation.
