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. 2021 Nov 23;221(1):e202105092. doi: 10.1083/jcb.202105092

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© 2021 Shen et al.

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Figure S1. — LUBAC is essential for ciliogenesis in mIMCD-3 cells and left–right asymmetry in zebrafish (related to Fig. 1). (A and B) HOIP was localized at the centrosome in interphase cells. HeLa cells (A) or RPE-1 cells (B) were transfected with GFP-HOIP and then stained with γ-tubulin (γ-tub, red). Insets show zoomed-in views of the boxed regions. Scale bars, 5 µm (main image) and 1 µm (magnified region). (C) RPE-1 cell lysates in Fig. 1 A were analyzed by immunoblotting with the indicated antibodies. (D) mIMCD-3 cells were transfected with control or LUBAC component siRNAs and serum starved for 48 h. The cells were stained with indicated antibodies. Scale bar, 1 µm. Ac-tubulin (Ac-tub) is a ciliary marker. (E) Quantification of the ciliated cells in D. (F) The knockdown efficiency of LUBAC in mIMCD-3 cells was analyzed by immunoblotting with the indicated antibodies. (G) RPE-1 cell lysates in Fig. 1 C were analyzed by immunoblotting with the indicated antibodies. (H) RPE-1 cells were transfected with control or LUBAC component siRNAs followed by serum starving for the indicated time points, and then the cells were stained with Ki-67, ARL13B, and Hoechst (DNA, blue). Scale bar, 10 µm. (I) Quantification of Ki-67–positive cells in H at the indicated time points. (J) The knockdown efficiency of rnf31-aMO was validated by the EGFP intensity expressed from the pCS2-UTR-rnf31-EGFP plasmid with aMO target sequence. Scale bar, 1 mm. (K) The knockdown efficiency of rnf31-sMO was analyzed by sequencing and RT-PCR. (L) The target sequence of rnf31-aMO and sequence of rnf31-rezmRNA. (M) The injection of rnf31-rezmRNA rescued curved body and pericardial edema in rnf31-aMO knockdown morphants. (N) The expression of rnf31-rezmRNA rescued the defective left–right asymmetry in rnf31-aMO knockdown morphants. Data are presented as mean ± SD of three independent experiments in E, I, M, and N. ***, P < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test in E and Bonferroni’s multiple comparisons test in M and N. The asterisks in C and F indicate nonspecific bands. Ctrl, control.