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. 2010 May 17;189(4):725–738. doi: 10.1083/jcb.201002047

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© 2010 Rodriguez-Fraticelli et al.

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Figure 2. — The siRNA-resistant form of ITSN2, vhITSN2, rescues normal lumen formation. (A) vhITSN2 is apically localized in MDCK cysts. Cells expressing vhITSN2 (green) were plated to form cysts for 72 h. Cysts were stained with gp135 and β-catenin (top). (B) ITSN2 colocalizes with pericentrin at centrosomes in interphase MDCK cells. Cells expressing vhITSN2 (green; left) were stained with pericentrin (centrosomal marker; middle) and acetylated tubulin (Acet Tub; right with DIC and merge). (C) ITSN2 localizes at spindle poles in mitotic MDCK cells. Cells expressing vhITSN2 (green; left) were stained with acetylated tubulin (AceTub; middle) and chromatin (blue; right and merge). (A–C) Bottom panels show the magnification image of the boxed areas indicated in the top panels. Arrows indicate ITSN2 localization. (D) Down-regulation of ITSN2 by siRNA in cells stably expressing vhITSN2. MDCK cells stable expressing vhITSN2 were transfected with siRNA to ITSN2 pool or with control siRNA and allowed to form cysts for 72 h. Total lysates were blotted for ITSN2 and α-tubulin. (E) Confocal microscopy images of the rescue effect of vhITSN2 in cells silenced for ITSN2 on lumen formation. Cells stably expressing vhITSN2 were transfected with ITSN2 pool or control siRNAs and plated to form cysts for 72 h. Cells were stained to detect gp135, vhITSN2, and β-catenin (βcat). (F) Quantification of cysts with normal lumens in cells expressing vhITSN2 and transfected with the control or ITSN2 pool siRNAs. Values are mean ± SD from three different experiments (n ≥ 100 cysts/experiment; *, P < 0.005). (G) Endogenous ITSN2 is present in centrosome-enriched fractions. MDCK cells were plated to reach confluence and then treated with 0.3 µM nocodazole and 1 µg/ml cytochalasin D for 4 h. A rapid isolation of centrosomes was performed using Ficoll gradient centrifugation of lysates, and then, centrosomes in the F2 fraction were precipitated by ultracentrifugation. The F2 supernatant (S) and pellet (P) were loaded together with fractions F3, F4, and F5, containing cytosolic and membrane proteins. The fractions were immunoblotted for ITSN2 and centrosomal, cytosolic, and membrane markers. (D and G) Molecular mass is indicated in kilodaltons. (H) Densitometry quantification of fractional enrichment represented as log₁₀(F2_P/mean_F3–5). Values are mean ± SD from three different experiments. PCNT, pericentrin. Bars, 5 µm.