Figure 1: The time course of ciliogenesis and cilium elongation induced by actin destabilization.

(a) Immunofluorescence images of RPE1 cells treated with DMSO or cytochalasin D (CytoD; 200 nM) for 16 h in the presence of 10% fetal bovine serum (FBS). Cilia were double-stained with antibodies specific for poly-glutamylated Tubulin (glu-TUB) and ARL13B. Blue channel was used for 4,6-diamidino-2-phenylindole (DAPI) staining. (b) Line graph of ciliated cell frequency in RPE1 cells plated in two different densities and treated with CytoD for the indicated times. Seeding density: 40 cells per mm² (low density), 160 cells per mm² (high density). See also Supplementary Fig. 1b. Error bars are s.e.m. (n=3 independent experiments). (c) Time-lapse imaging of a live RPE1 cell stably expressing Smo-EGFP (RPE1-Smo-EGFP) for up to 3 h after CytoD treatment. (d) Graph showing the changes of ciliary length of RPE1-Smo-EGFP cell treated with DMSO or CytoD from time-lapse microscopic images (n=20 cells for each treatment). Smo-EGFP fluorescence was used for the measurement of cilium length. (e) Immunofluorescence images of RPE1 cells treated with DMSO or CytoD for 18 h in the early S phase arrest with a single thymidine block (36 h before fixation). Cilia were stained with anti-glu-TUB antibody. (f) Quantification of experiments illustrated in e. Error bars are s.d. (n=2 independent experiments). Scale bars in a,e are 20 μm, and c is 5 μm.