Journal: Cell Death and Differentiation

Article Title: BimEL is phosphorylated at mitosis by Aurora A and targeted for degradation by β TrCP1

doi: 10.1038/cdd.2013.93

Figure Lengend Snippet: Knockdown of Bim protects cells from apoptosis induced by Aurora A inhibition. (a) Bim expression was knocked down in HeLa cells using two different lentivirus delivered shRNAs (sh-Bim1 and shBim2). A non-silencing shRNA (sh-NS) was used as control. (Top) Knockdown and control cells were treated with 5 μM MLN8054 for 72 h. Resulting tissue culture plates were stained with crystal violet. (Bottom) Immunoblots of whole-cell extracts confirming knockdown in Bim shRNA-expressing cells. β-Actin is shown as loading control. (b) Quantitation of cell numbers from experiment in a. Student's t-test was used to determine statistical significance (*P≤0.05, **P≤0.01). (c) Control (sh-NS) or sh-Bim1 and sh-Bim2 HeLa cells were treated with 5 μM MLN8054 for 48 h. Immunoblots of whole-cell extracts were performed to confirm Bim knockdown and cleaved PARP-1 and Caspase 3 were used to demonstrate apoptotic effect of MLN8054. β-Actin is shown as a loading control. (d) HeLa cells were treated as described for c, stained with AnnexinV and 7AAD, and analyzed by flow cytometry. The percentage of AnnexinV-positive cells is indicated for each treatment. Grey bars indicate untreated and black bars are treatment with MLN8054

Article Snippet: Cells were collected by scraping and washed twice with ice-cold PBS then resupended in 100 μ l of AnnexinV binding buffer (2.5 mM CaCl 2 , 140 mM NaCl, 7.75 mM HEPES (pH 7.4)) and then incubated with PE-AnnexinV (BD Biosciences) and 7-amino-actinomycin D (7AAD; A.G. Scientific, San Diego, CA, USA) according to the manufacturing protocol.

Techniques: Inhibition, Expressing, shRNA, Staining, Western Blot, Quantitation Assay, Flow Cytometry