Figure 2.

Treatment with forskolin results in a rescue of cilia biogenesis and elongation defects but not of ciliary localisation of ARL13B in JBTS5 cells. (a) Immunofluorescence microscopy of control fibroblast lines C1, C2 and JBTS5 lines P1, P2. Untreated, serum starved cells (left panel) and cells treated with 25 μM forskolin (right panel). Treatment with forskolin results in an increase of ciliated cells upon serum starvation and in a rescue of ciliary phenotype in JBTS5 cells. DAPI- blue (Nuclei), Green - ARL13B, Red - Acetylated α-tubulin. Scale bar 20 μm. (b) Quantification of cilia length in control fibroblast lines C1, C2 and JBTS5 lines P1, P2, untreated or treated with forskolin. Mean length of JBTS5 fibroblast cilia decreased from 6.7 μm to 5.5 μm and 8.0 μm to 4.8 μm in P1 and P2, respectively, after treatment with forskolin. *P < 0.05, **P < 0.01, ***P < 0.001, ns, non-significant, one-way ANOVA, n = 341. (c) Quantification of percentage of ciliated cells in control fibroblast lines C1, C2 and JBTS5 lines P1, P2, untreated or treated with forskolin. Percentage of ciliated JBTS5 fibroblasts increased from 34% to 59% and from 45% to 71% in P1 and P2, respectively, after treatment with forskolin. ***P < 0.001, ns, non-significant, Fisher’s exact test. Bonferroni correction was used to adjust for multiple comparisons, n = 1244. (d) Quantification of ciliary ARL13B intensity normalised to the background in control fibroblast lines C1 and JBTS5 lines P1 and P2, untreated or treated with forskolin. Values are expressed as ARL13B ciliary intensity relative to ciliary intensity in C1 cells. ***P < 0.001, ns, non-significant, one-way ANOVA, n = 239. FOR, forskolin.