Journal: PLoS ONE
Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1
Figure Lengend Snippet: Cyclin B1 and Securin are degraded during mitotic slippage induced by inhibition of transcription and translation. A) Analysis of Cyclin B1, Securin and CDK1 levels by western blotting. NIH3T3 cells were incubated with nocodazole (noc) for 14 h and treated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 35.5 µM) with or without the inhibitor of the proteasome MG132 (25 µM), for 4 h; β-actin was used as loading control. B) Quantification of Cyclin B1 and Securin proteins in noc-, noc-ActD-, noc-ActD-MG, noc-CHX and noc-CHX-MG-cells. Normalization of Cyclin B1 and Securin was calculated on the ratio of these proteins per β-actin protein present in each condition. Data presented as mean ± S.D. from three independent experiments. C and D) Immunofluorescence microscopy: asynchronous population (control) and noc-cells treated with ActD for 4 h; DAPI stains the DNA (blue) and Cyclin B1 (C) and Securin (D) are stained in green. Arrowheads point mitotic cells and arrows point cell that undergone mitotic slippage. The adapted cells shown are representative of both ActD and CHX induced mitotic slippage. Scale bar = 25 µm.
Article Snippet: NIH3T3 cells were incubated for 14 h with nocodazole and further treated for 4 h with either 35.5 µM of cycloheximide (CHX), a de novo protein synthesis inhibitor , or 8 µM of actinomycin D (ActD), an inhibitor of transcription .
Techniques: Inhibition, Western Blot, Incubation, Immunofluorescence, Microscopy, Staining