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. 2019 Nov 14;132(22):jcs233973. doi: 10.1242/jcs.233973

Fig. 1.

Fig. 1.

MICAL-L1 regulates ciliogenesis. (A–E) RPE-1 cells were mock-treated (with Lipofectamine in the absence of the siRNA oligonucleotide), treated with MICAL-L1 siRNA or with EHD1 siRNA for 72 h. During the last 48 h, the cells were serum-starved to induce ciliogenesis. Cilia were detected by immunostaining with an anti-acetylated tubulin antibody (marked by yellow arrows in A–C). Compared to mock-treated cells (A), fewer cilia were generated upon MICAL-L1 depletion (B), and upon EHD1 depletion (C). MICAL-L1 and EHD1 knockdown efficacies (>90%) were verified by immunoblotting (D). The percentage of ciliated cells following serum-starvation was quantified and presented as a bar graph (E). Scale bar: 10 μm. (F–K) RPE-1 (F–H) or NIH3T3 (I–K) cells were fixed or treated with medium containing 0.2% serum for 48 h. Ciliated (F,G,I,J) or non-ciliated cells (H,K) were immunostained with antibodies to MICAL-L1 (red) and acetylated tubulin (green). MICAL-L1 localized to the cilia (F,I) and the basal body (G,J) upon serum starvation, and the centrosome (H,K) even in the absence of ciliogenesis. Micrographs in F–H were obtained by sr-SIM, whereas micrographs in I–K were from confocal microscopy. Scale bar: 0.5 μm. (L–P) RPE-1 cells were mock-treated (M), or subjected to MICAL-L1 depletion (N) or syndapin 2 siRNA-mediated depletion (O), followed by serum starvation for 24 h, and immunostained with acetylated tubulin (red) and CP110 (green) antibodies. CP110 removal from the m-centriole was impaired upon MICAL-L1 depletion (N; green dots co-localized with red acetylated tubulin; quantified in P). Upon syndapin 2 depletion, CP110 was released from the m-centriole (O, quantified in P). The efficacy of syndapin 2 knockdown was determined by immunoblotting (L). Graph shows the percentage of cells displaying CP110 removal from the m-centriole (P). Scale bar: 0.5 μm. *P<0.001; ns, not significant (P>0.05), for comparison between mock- and siRNA-treated cells; n=3 experiments (>100 cells quantified for each experiment). Error bars denote standard deviation.