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. Author manuscript; available in PMC: 2011 Aug 20.
Published in final edited form as: Cell. 2010 Aug 12;142(4):625–636. doi: 10.1016/j.cell.2010.07.019

Figure 5. Alternative splicing regulation is coupled to cell cycle control.

Figure 5

(A) Screen hits span an AURKA-centered protein interaction network. Circles (‘nodes’) depict proteins, connected by lines (‘edges’) representing validated protein-protein interactions. A maximum of two non-hit bridges was permitted to connect hits to AURKA, as described in Supplemental Data.

(B) Drugs disrupting the cell-cycle promote pro-apoptotic Bcl-x AS. Bcl-x reporter cells were treated with nocodazole, aurora inhibitors VX-680 or ZM447439, or staurosporine for 18 hours. 3-fold dilution series were tested with the following maximum concentrations: 200 nM nocodazole, 10 μM VX-680, 30 μM ZM447439, 33nM staurosporine. %-Venus-positive values for drug treatments were normalized to DMSO-treatments; plotted values reflect means of 8–12 measurements +/− SD. Nocodazole, VX-680, and ZM447439, but not staurosporine, caused significant pro-apoptotic shifts in Bcl-x splicing (p<0.001). LD50 ranges derived from cell counts are shown below the x-axis. Wells with >33nM staurosporine had >95% death, so they were not quantified (see Figure S5).

(C) Aurora inhibitors induced mitotic arrest coupled with pro-apoptotic Bcl-x splicing. Bcl-x reporter cells were treated with 3 μM VX-680, 10 μM ZM447439 or DMSO, stained with propidium iodide, and analyzed for DNA content and Venus fluorescence by flow cytometry.

(D) Asynchronous, actively cycling cells from (C) show little to no Bcl-x splicing fluctuations relative to arrested cells.

(E) S-phase arrest by double thymidine block did not alter Bcl-x splicing, indicating specificity for mitotic arrest. Cells were analyzed as in (C).

(F) Bcl-x splicing regulation precedes mitotic arrest. Asynchronous or thymidine-synchronized cells from (E) were treated with the indicated inhibitors or DMSO. Cells were analyzed by automated microscopy 16 hours after thymidine release, precluding an intervening round of mitosis for synchronized cells. The response in pre-synchronized cells confirms a splicing shift upstream of mitotic arrest.