Fig. 3

IFT140 variant rescue analysis in mIMCD3-Ift140^−/− cells. CRISPR/Cas9-derived Ift140 knockout mIMCD3 (mIMCD3-Ift140^−/−) cells were transfected with wild-type, p.Tyr923Asp or p.Tyr332Valfs*18 mutant IFT140-Myc-DDK constructs and the cilium rescue abilities were assessed. Immunofluorescent images are representative for the measurements showing the base of the cilium using RPGRIP1L (pink), an antibody targeting FLAG to visualize the IFT140-Myc-DDK construct (red), and IFT88 in green. Scale bar represents 2 µm. a Immunoblot analysis of IFT140 protein levels in CRISPR/Cas9 negative control mIMCD3 cells and CRISPR/Cas9-derived Ift140 knockout mIMCD3 cells in the absence or presence of the IFT140-Myc-DDK construct. The immunoblot was stained with an antibody targeting IFT140. b Ciliogenesis measurements in untreated mIMCD3-Ift140^−/− cells and after transfection with wild-type or mutant IFT140-Myc-DDK constructs. Untreated mIMCD3-Ift140^−/− cells showed < 1% ciliogenesis. Transfection with the wild-type IFT140 construct rescued ciliogenesis in 11% of the analyzed cells, while both mutant IFT140 constructs, p.Tyr923Asp and p.Tyr332Valfs*18, rescued ciliogenesis in 6% of the cells (N = 3 with > 250 cells analyzed per experiment). c Length measurements of the rescued cilia based on the IFT88 staining of the cilium. The dots represent individual cells and the horizontal line indicates the mean (N ≥ 75). Significance was measured using the unpaired two-tailed t test; wild-type compared to either mutant showed a p < 0.0001. d Ciliary localization of the IFT-B protein IFT88. There is a significant increase of cilia displaying a ciliary tip accumulation of IFT88 in both the p.Tyr923Asp (50%) and the p.Y332Vfs*18 (56%) mutant IFT140 constructs compared to wild-type (23%) (Fisher’s exact two-tailed test showed a p < 0.0001 when comparing either mutant to wild-type IFT140). N = 3 with > 50–100 cells per experiment