Fig. 4: RP13- RPE cilia have increased length, abnormal morphology and altered transition zone structure. | Nature Communications

Fig. 4: RP13- RPE cilia have increased length, abnormal morphology and altered transition zone structure.

From: PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

Fig. 4

A PRPF8 localisation to proximal ciliary membrane (ARL13B) in RP13 and RP13-Cas9-RPE cells, scale bar 10 μm; (B) Bar chart of cilia incidence, n = 15 fields of view from 3 donors and controls; (C) Boxplot of cilia length, n = 3901 and 3630 for RP13 (median = 2.96 μm, LQ = 2.36 μm, UQ = 3.63 μm, min = 1.09 μm, max = 7.72 μm, n = 3901) and RP13-Cas9 (median = 2.66 μm, LQ = 2.19 μm, UQ = 3.24 μm, min = 1.10 μm, max = 7.89 μm, n = 3630), p = 2.4 × 10−53, respectively where n is the total number of cilia from 3 donors and controls; (D) Representative image of a normal cilium, scale bar 1 μm. Blue arrowhead indicates “tail-like” morphology. Bar graph quantifies the percentage of normal cilia, n = 1499 and 1986 cilia analysed in Cas9 and RP13-RPE cells respectively (p = 0.0099); (E) Representative image of cilium without proximal ARL13B localisation, scale bar 1 μm. Bar graph quantifies the percentage of cilia without this localisation in RP13 and RP13-Cas9-RPE; n = 15 fields of view from 3 donors and controls (p = 0.0033); (F) Representative image and quantification of abnormal “blob”-shaped cilium, scale bar 1 μm; n = 15 fields of view from 3 donors (p = 0.00049); (G) Ciliary volume and length quantified using SBF-SEM, n = 184 individual cilia from 3 donors and controls, scale bar 1 μm; (H) iPSC-RPE cilia labelled for polyglutamylated tubulin (monoclonal antibody GT335), scale bar is 1 μm. The white arrowhead indicates the mother centriole (MC), and the purple arrowhead indicates the daughter centriole (DC, in RP13-RPE only). The yellow line indicates the ciliary transition zone (TZ). Boxplot quantifies average TZ length in RP13 (median = 0.40 μm, LQ = 0.35 μm, UQ = 0.46 μm, min = 0.24 μm, max = 0.59 μm, n = 27) and RP13-Cas9 (median = 0.29 μm, LQ = 0.21 μm, UQ = 0.36 μm, min = 0.12 μm, max =  0.57 μm, n = 36) iPSC-RPE cilia; n is number of cilia (p = 1.25 × 10−6); (I) ARL13B and CEP290 localisation, with bar graph expressing co-localisation as Pearson’s correlation coefficient, n = 15 fields of view obtained from 3 donors and controls (p = 0.0031); scale bar 400 nm; (J) Representative GT335 and IFT88 localisation using super-resolution microscopy, scale bar 1 µm. Bar graph quantifies mean IFT88 fluorescence intensity, n = 15 fields of view obtained from 3 donors and controls (p = 0.00012). B, C, E–J results are mean ± SEM; statistical significance analysed by two-tailed Student’s t-test with Welch’s correction (*P-value 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001.

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