Figure 1.

The linear ubiquitin ligase complex LUBAC is required for ciliogenesis in human cells and zebrafish. (A) RPE-1 cells were transfected with control or LUBAC component siRNAs. The cells were serum starved for 48 h and stained with the ciliary marker Ac-tubulin (Ac-tub) and γ-tubulin (γ-tub). Scale bar, 1 µm. (B) Effects of LUBAC depletion on ciliogenesis in the absence of serum in RPE-1 cells. Quantification of the ciliated cells in A. (C) The mCherry-tagged, RNAi-resistant form of WT HOIP, but not C885S mutant, rescued the defects of ciliogenesis in HOIP-depleted RPE-1 cells. Quantification of the ciliated cells in mCherry-positive RPE-1 cells. (D) Knockdown of rnf31 (aMO and sMO) in zebrafish caused the curved body and pericardial edema at 48 hpf. Scale bars, 1 mm. The arrows mark curved body, and arrowheads mark pericardial edema. (E) Quantification of curved body and pericardial edema in D. (F) rnf31-knockdown morphants displayed left–right asymmetry defects. The cmlc2 probe was used to label the heart loop in the whole-mount in situ hybridization at 26 hpf. Scale bar, 200 µm. (G) Quantification of left–right asymmetry in F. (H) Knockdown of rnf31 (aMO and sMO) impaired ciliogenesis in KV at 10 somite stage (10 s). Scale bar, 10 µm. (I) Quantification of the cilia number of KV in H. Each dot represents one fish. Data are presented as mean ± SD of three independent experiments in B, C, E, G, and I. ***, P < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test in B, E, G, and I and Bonferroni’s multiple comparisons test in C. Ctrl, control.