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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 1. — ITSN2 silencing abrogates lumen formation and reduces GTP-bound Cdc42 levels. (A) Down-regulation of ITSN2 by siRNA. MDCK cells were transfected with siRNA ITSN2-1, ITSN2-2, ITSN2-3, and a pooled combination of all three or with control siRNA and allowed to form cysts for 72 h. Total lysates were blotted for ITSN2 and tubulin. (B) Confocal microscopy images of the effect of ITSN2 siRNA–mediated silencing on lumen formation. Cells were transfected with ITSN2 siRNA pool or control siRNA and plated to form cysts for 72 h. Cells were stained to detect gp135, β-catenin (βcat), and ZO-1. (C) Quantification of cysts with normal lumens in cells transfected with control siRNA, Cdc42 siRNA, or ITSN2 siRNA pool. Values are mean ± SD from five different experiments (n = 100 cysts/experiment; *, P < 0.05; **, P < 0.005). (D) siRNA-mediated silencing of ITSN2 inhibits activation of Cdc42. Cells were transfected with ITSN2 siRNA pool, Cdc42 siRNA, or control siRNA. Extracts from these cells were pulled down with Pak-PBD-GST. Total and GTP-bound Cdc42 was detected by immunoblotting with specific antibodies to Cdc42 or tubulin. PD, pull-down; WB, Western blot. (E) Quantification of Cdc42-GTP levels in ITSN2-silenced cells. The ratios of GTP-bound to total protein were calculated relative to tubulin content. Values shown are mean ± SD from three different experiments (*, P < 0.005). (F) Down-regulation of ITSN2 by siRNA in cells with the inducible expression of Cdc42V12-myc. MDCK cells stable expressing Cdc42V12-myc under the control of the tet-off repressor were transfected with siRNA to ITSN2 pool or Cdc42 or with control siRNA and allowed to form cysts for 72 h in the presence of Dox (Cdc42V12-myc expression repressed) or not (induced). Total lysates were Western blotted for Cdc42 and α-tubulin. (A, D, and F) Molecular mass is indicated in kilodaltons. (G) Confocal microscopy images of the rescue effect of Cdc42V12-myc in cells silenced for ITSN2, Cdc42, or control on lumen formation. Cells were transfected with ITSN2 pool, control, or Cdc42 siRNAs and plated to form cysts for 72 h in the presence (Cdc42V12 repressed) or the absence (induced) of 20 µmol Dox. Cells were stained to detect actin, Cdc42V12-myc, and β-catenin. (H) Quantification of cysts with normal lumens in cells expressing or not Cdc42V12-myc and transfected with the control, Cdc42, or ITSN2 pool siRNAs. Values are mean ± SD from three different experiments (n ≥ 100 cysts/experiment; *, P < 0.005). Bars, 5 µm.