Fig. 6
PRPF31 loss causes defects in cilia incidence and structural organisation. a PRPF31 siRNA knockdown in human hTERT-RPE1 cells causes a significant decrease in cilia incidence (lower left) and PRPF31 protein levels (lower right) compared to scrambled negative control (siScr) siRNA, scale bar: 10 μm; b Gli1 reporter assays of Shh activity measured in NIH3T3-GL cells following knockdown for Ptch1 (positive control), scrambled negative control siRNA (siScr) and Prpf31. Cells were treated with either 100 nM SAG or vehicle control for 48 h, as indicated. Assays results are expressed in arbitrary units of the ratio of firefly: Renilla luciferase activities; c Ciliary localisation of IFT88 (green) in primary cilia of hTERT-RPE1 cells (visualised by staining for γ-tubulin and poly-glutamylated tubulin; red) showing mislocalisation of IFT88 (arrowheads) at ciliary tips following PRPF31 knockdown. Bar graph quantitates the percentage of cilia with IFT88 at their tip. Scale bar: 1 μm; d Visualisation and quantitative analysis of the transition zone protein CC2D2A (green) and ARL13B (red); e Visualisation and quantitative analysis of the transition zone (TZ) protein RPGRIP1L (green) and cilia (γ-tubulin and poly-glutamylated tubulin; red) showing mislocalisation of RPGRIP1L from the TZ into the ciliary axoneme (arrowheads) following PRPF31 knockdown. f, g Ciliary localisation of IFT88 and RPGRIP1L (green) in RP11 - RPE cells showing mislocalisation of IFT88 (arrowheads) at ciliary tips and RPGRIP1L from the TZ into the ciliary axoneme (arrowheads). a–g Data shown as mean ± SEM, n = 3. Statistical significance of pairwise comparisons is indicated by n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s unpaired t test). c–f Scale bar: 1 μm
