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. 2011 Dec 1;22(23):4549–4562. doi: 10.1091/mbc.E11-05-0405

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© 2011 Baron Gaillard et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Hook2 depletion hinders ciliogenesis without Golgi breakdown in ARPE 19 cells. (A) A mix of 3 siRNAs (H1, H2, and H3) targeting human Hook2 mRNA was used to transiently decrease Hook2 expression for 7 d as measured by WB analysis of Hook2 levels in ARPE19 total cell lysates treated either with Hook2 siRNAs or luciferase siRNA (control siRNA). Antibodies against α-tubulin and GAPDH were used as loading controls. Molecular weight (MW) markers are indicated on the left in kilodaltons. (B) Quantification of Hook2 depletion. Error bars, SD; n = 20 and p < 0001. (C) SiRNA Hook2-depleted cells were fixed in PFA and compared with control siRNA-treated cells for their ability to promote ciliogenesis (acetylated tubulin staining) and maintain Golgi architecture (TGN46 staining) by immunofluorescence and confocal microscopy analysis. Bar, 10 μm; inset magnification, ×15. Asterisks indicate cilia. Note that Hook2 siRNA depletion does not induce breakdown but rather a compaction of the Golgi apparatus. (D) Quantification of ciliogenesis inhibition by Hook2 depletion from three independent experiments. Error bars, SD; n = 200 cells per condition and p < 0001. (E) Quantification of Golgi compaction by Hook2 depletion from three independent experiments. Error bars, SD; n = 100 cells per condition and p < 0001. (F) SEM analysis of control and Hook2 siRNA transiently depleted ARPE19 cells. Bar, 5 μm; inset magnification, ×2. Arrows indicate cilia in the control cells, and arrowheads mark some microvilli in Hook2-depleted cells. See also Supplemental Figure S2.