Fig. 2. Wrch-1 depletion increases focal adhesions. (A) Fluorescence micrographs of Wrch-1-depleted cells and control cells. Cells were transfected with siRNA targeting luciferase (control) or Wrch-1 (W-1 and W-2) as described in Materials and Methods. Twenty-four hours after transfection, cells were re-plated onto laminin-coated coverslips for another 48 hours. Subsequently, cells were fixed and processed for indirect immunofluorescence using a anti-vinculin antibody and a Texas-Red-labeled secondary antibody, and co-stained for F-actin (FITC-phalloidin). Bar, 10 µm. (B) Quantitative PCR analysis of Wrch-1 knockdown. Cells were transfected as described for A. Seventy-two hours after transfection, total RNA was isolated and analyzed for Wrch-1 mRNA levels. Shown is the average of seven independent experiments ± s.e.m. (C) Wrch-1 depletion diminishes cell spreading. Cells were processed as described for A. For each treatment, the average cell area and s.e.m. was determined for 20 to 25 cells using Esee software (Inovision). Data shown are representative of three independent experiments. (D-F) Quantification of focal adhesions (D), average focal adhesion length (E) and focal adhesion brightness (F) was performed as described in Materials and Methods. For each treatment, the average number of focal adhesions per cell, the average focal adhesion length and brightness (± s.e.m.) was determined for eight cells, randomly selected. Data shown are representative of three independent experiments. ^*P<0.05, two-tailed t-test.