Figure 7.

Cilia length and U12 intron splicing are restored in cells derived from an affected individual upon reintroduction of SCNM1

(A) Representative immunofluorescence images showing normal cilia length in fibroblasts derived from P2 (P) retrotansduced with SCNM1 but not in P2 fibroblasts transduced with the empty viral vector (pBABE). No significant differences were observed in similarly retrotransduced normal control C2 (C) cells included in the same assay. Green [acetylated tubulin (AcTub) + gamma tubulin (γ-Tub)]: cilia; blue (DAPI): nuclei. Scale bars: 10 μm.

(B) Graphs illustrating cilia length, distribution of cilia length, and frequency of ciliated cells in control (C) and affected individual (P) primary fibroblasts retrotransduced with SCNM1 or the empty vector (pBABE). For each cell line, the length of a minimum of 120 cilia was measured (n = 3 [C], n = 4 [P]), and at least 125 cells were analyzed to calculate the ratio of ciliated cells (n = 3 [C], n = 3 [P]). Data are presented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Kruskal-Wallis test with Dunn’s multiple comparison test and one-way ANOVA with Bonferroni’s post-hoc test for comparison of selected pairs were used for cilia length and ciliation frequency, respectively.

(C–G) Relative quantification of TMEM107 (C), FAM92A (D), ZC3H8 (E), DERL2 (F), and C17orf75 (G) mRNA levels by RT-qPCR in control (C) and affected individual (P) cultured primary fibroblasts retrotransduced with SCNM1 or the empty retroviral vector (pBABE). RNA samples used were isolated from DMSO-treated cultures. Scatterplots show mean ± SD (n = 3 [C], n = 4 [P]). Gene expression for TMEM107, FAM92A, ZC3H8, and DERL2 was normalized against the geometric mean of GAPDH and GUSB mRNA levels. For C17orf75, GUSB expression was used for normalization. The ΔCt mean value of control cells (C) retrotransduced with pBABE was used as calibrator sample. ^∗∗∗p < 0.001. One-way ANOVA with Tukey’s multiple comparison test.

(H–K) Representative immunoblots of FAM92A (H), ZC3H8 (I), DERL2 (J), and C17orf75 (K) showing reestablishment of the normal levels of these proteins in affected individual (P) primary fibroblasts after SCNM1 retroviral delivery. Control fibroblasts are labelled as C. Blots were conducted in cell extracts from DMSO (−)- and SAG (+)-treated cells. (H and I): n = 3 (C), n = 4 (P); (J and K): n = 2 (C and P). In (I), arrowheads mark WT (ZC3H8) and truncated (ZC3H8Δ) ZC3H8 isoforms. In (K), the asterisk designates a non-specific band. Tubulin (TUB) and vinculin (VINC) served as a loading control.