Figure 7.

Treatment with Ex41 skip ASO rescues cilia length and biogenesis in JBTS5 fibroblasts. (a) Representative immunofluorescence images of control fibroblasts C2 and JBTS5 cells P2, treated with 5 μM Std ASO or 5 μM Ex41 skip ASO and serum starved for 48 h. Treatment with Ex41 skip ASO results in a rescue of ciliary phenotype in JBTS5 cells. Green - ARL13B, Red – acetylated α-tubulin, Violet - pericentrin. Scale bar 3 μm. (b) Quantification of cilia length in control fibroblast lines C1, C2 and JBTS5 lines P1, P2, treated with 5 μM Std ASO or 5 μM Ex41 skip ASO. The mean cilia length measured for C1 was 5.3 μm when treated with Std ASO and 5.6 μm after treatment with Ex41 skip ASO. The mean cilia length measured for C2 was 5.6 μm when treated with Std ASO or Ex41 skip ASO. Mean length of JBTS5 fibroblasts P1 cilia decreased from 7.0 μm to a near-control length of 4.9 μm after treatment with Ex41 skip ASO. Similarly, JBTS5 fibroblasts P2 mean cilia length decreased from 7.6 μm to near-control length of 5.7 μm following treatment with Ex41 skip ASO. ***P < 0.001, ns, non-significant, one-way ANOVA, n = 1168, data from three independent experiments. (c) Quantification of percentage of ciliated cells in control fibroblast lines C1, C2 and JBTS5 lines P1, P2, treated with 5 μM Std ASO or 5 μM Ex41 skip ASO. The percentage of ciliated cells for C1 was 98% when treated with Std ASO and 71% after treatment with Ex41 skip ASO. The percentage of ciliated cells measured for C2 was 98% when treated with Std ASO and 91% after treatment with Ex41 skip ASO. Percentage of ciliated JBTS5 fibroblasts P1 increased from 24% to 57% after treatment with Ex41 skip ASO. Similarly, the percentage of ciliated JBTS5 fibroblasts P2 increased from 30% to 69%, following treatment with Ex41 skip ASO. ***P < 0.001, ns, non-significant, Fisher’s exact test. Bonferroni correction was used to adjust for multiple comparisons, n = 910.