Figure 2.

Depletion of LUBAC specifically affects CP110–CEP97 complex removal during ciliogenesis. (A) A table summarizing the centrosome localization of cilia-associated proteins in LUBAC-depleted RPE-1 cells. (B–F) RPE-1 cells were transfected with control or LUBAC component siRNAs and serum starved for 30 min (D) or 48 h (B, C, E, and F). The cells were stained with indicated antibodies. Scale bar, 1 µm. (B) The localization of centrosomal protein γ-tubulin (γ-tub) was not affected by depletion of LUBAC. (C) The localization of the distal end appendage marker CEP164 was not disrupted by LUBAC depletion. (D) MYO5A was normally localized at the mother centriole in LUBAC-depleted cells. (E and F) Depletion of LUBAC affected the removal of CP110 and CEP97 from the mother centrioles. (G) Knockdown of CP110 rescued the defective ciliogenesis in HOIP-depleted cells. RPE-1 cells were transfected with HOIP or CP110 siRNAs or cotransfected with HOIP and CP110 siRNAs Cells then were serum starved for 48 h and stained with ARL13B (red). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 2 µm (magnified region). (H) Quantification of the ciliated cells in G. (I) Immunoblot of the RPE-1 cell lysates in G with the indicated antibodies. Data are presented as mean ± SD of three independent experiments in B–F and H. ***, P < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test in B–F and Bonferroni’s multiple comparisons test in H. Ctrl, control.