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. 2019 Aug 29;294(42):15418–15434. doi: 10.1074/jbc.RA119.008615

Figure 2.

Figure 2.

C7orf43 is required for ciliogenesis in human cells and zebrafish embryos. A, Western blot analysis of lysates from RPE-1 cells treated with control and C7orf43 siRNAs for 72 h. C7orf43 and β-actin antibodies were used for immunoblotting. Protein levels of C7orf43 compared with siControl were determined by densitometry analysis and are shown below blots normalized for differences in actin expression between RNAi treatments. Representative results from three independent experiments are shown. B and C, quantification of ciliation in NeoHDF (B) and RPE-1 (C) cells treated with C7orf43 siRNAs as in A and serum-starved for the final 24 h. Cells were stained with acetylated tubulin (Actub) and pericentrin antibodies and 4′,6-diamidino-2-phenylindole and imaged by epifluorescence microscopy. Representative images (right) are shown in B. Scale bar, 10 μm. Cells were counted for NeoHDF, siControl (n = 607) and siC7orf43 (n = 521) (B). Cells were counted for RPE-1, siControl (n = 1287), and siC7orf43 (n = 1070) (C). Means ± S.E. (error bars) are shown from three independent experiments. Two-tailed, unpaired Student's t test was used. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, Western blot analysis of lysates from 2-day postfertilization (dpf) zebrafish embryos injected with c7orf43 MO and probed with C7orf43 and β-actin antibodies. Representative results from two independent experiments are shown. E, panels show representative brightfield images of zebrafish embryos treated as in D from three or four independent experiments. Rescued embryos (right) were co-injected with both c7orf43 MO and human C7ORF43 RNA (hC7orf43). F–H, top, immunostaining of ciliated organs from 2-dpf embryos treated as in D showing ciliation. White arrowheads, neuromasts cilia. Bottom, quantification of organs with abnormal cilia in 2-dpf embryos. n > 14 olfactory vesicles; n > 27 neuromasts; n > 10 olfactory placodes. Note that ciliation was rescued upon injection of human C7orf43 mRNA, validating MO specificity. Two-tailed, unpaired Student's t test was used. Means ± S.E. are shown from three or four independent experiments. **, p < 0.01.