Depletion of CHC17 clathrin amplifies mitotic centrosome number and induces multipolar spindle formation. (A) HeLa-GFP–α-tubulin cells were treated (72 h) with siRNA-targeting CHC17, CLCs LCa and LCb (siCLC), CHC22, both CHC17 and CLC, or with nonsilencing siRNA (siNonsilencing). Cell lysates were analyzed for protein depletion by immunoblotting for proteins at the left. β-actin serves as a loading control. Molecular mass marker position is shown right. (B and C) HeLa-GFP–α-tubulin cells were treated with siRNA, as in A, and processed for IF to detect γ-tubulin and CLC or CHC22. GFP–α-tubulin was visualized in green. Red-green merge is yellow. Bars, 7.5 µm. (insets) Boxes in each frame are magnified threefold. Bars, 2.0 µm. All images are 2D. (D) The percentage of HeLa-GFP–α-tubulin cells with more than two centrosomes (shaded bars) and the percentage of cells with more than two spindle poles (open bars) were quantified for ≥200 mitotic cells in prometaphase or metaphase for each siRNA treatment. Centrosomes were identified by γ-tubulin staining at spindle poles. Occasional spindle poles without obvious centrosomes were observed, so these were scored separately. Data represent the mean ± SD of four to five independent experiments. Statistically significant results are shown in comparison with nonsilencing siRNA conditions (*, P < 0.1 based on a one-way analysis of variance Friedman’s matched pairs test with Dunn’s multiple comparison post-hoc test).