Control of Centrin Stability by Aurora A Figure 4. S170D-centrin drives centrosome amplification. (A) Constant expression of S170D-centrin drives centrosome amplification in HeLa Tet-On S170D-centrin cells treated continuously with doxycycline. Cells were stained with an antibody against HA (green) and a polyclonal antibody against centrin (red). Scale bar = 10 microns. (B) Cells that were not induced with doxycycline have normal centrosomes. Cells were stained as in panel (A). Scale bar = 10 microns. (C–F) HeLa Tet-On HA-WT-centrin or HA-S170A-centrin cells transfected with GFP-WT Aurora A and allowed to recover for 24 hours. Cells were then transfected with control shRNA or centrin shRNA and treated with doxycycline to induce HA-centrin expression. Aurora A expression was noted by GFP positivity and cells were stained with centrin (red) and gamma-tubulin (turquoise). A vector control Western blot is shown in Figure S3. Cells over-expressing HA-wildtype centrin in the presence of endogenous centrin have large, numerous centrosomes (C) in virtually all cells. Cells over-expressing HA-wildtype centrin in the absence of endogenous centrin have large, numerous centrosomes (D) in most cells. Conversely cells over-expressing HA-S170A centrin in the presence of endogenous centrin occasionally have slightly enlarged centrosomes (E) and cells over-expressing HA-S170A centrin in the absence of endogenous centrin rarely have slightly enlarged centrosomes (F). GFP positive cells were analyzed for centrosome amplification based on increased size and/or number of centrosomes. The number and percentage of cells with centrosome amplification is presented below each panel. Scale bar = 2.5 microns. (G–I) Frozen breast tissue including normal adjacent breast (G), ductal carcinoma in situ (H) and invasive breast cancers (I) were stained with antibodies directed against Aurora A (green), p-S170 centrin (turquoise), and gamma-tubulin (red). Cells were stained with DAPI to mark the DNA. Scale bar = 10 microns. doi:10.1371/journal.pone.0021291.g004 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21291