Figure S1. Alisertib and Barasertib Induces Apoptosis Following Mitotic Slippage

SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Alisertib and Barasertib induces apoptosis following mitotic slippage.

HeLa cells stably expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of Alisertib or Barasertib. Individual cells were then tracked for 38 h with time-lapse microscopy. Each horizontal bar represents one cell (n=50). Key: light grey=interphase; black=mitosis (from DNA condensation to anaphase or mitotic slippage); dark grey=interphase after mitotic slippage; truncated bars=cell death.

Figure S2. The identity of phosphorylated Aurora kinases during mitosis.

(A) AURKA and AURKB are phosphorylated during mitosis. HeLa cells were transfected with siRNAs against control, AURKA, or AURKB. The cells were synchronized with double thymidine block and released into medium containing nocodazole. The anti-phospho-Aurora kinase antibody recognized three bands. The top two bands co-migrated with AURKA and AURKB, respectively. The bottom band co-migrated with a band recognized by an antibody raised against AURKC, this band may not be the bona fide AURKC (see panel (B). Although phospho-AURKA and -AURKB could be depleted with specific siRNAs, the fastest-migrating band was also depleted by the AURKB siRNA. These data suggested that instead of AURKC, the bottom band might represent a phosphorylated form or an isoform of AURKB (37).

(B) The band recognized by AURKC antibody may not be bona fide AURKC. HeLa cells were transfected with siRNAs against AURKA, AURKB, or AURKC. Several concentrations of siRNA were used (10, 25, 50, 100 nM). After 36 h, lysates were prepared and the indicated proteins were detected with immunoblotting. Uniform loading was confirmed by immunoblotting for actin.

Figure S3. The Aurora B inhibitor ZM447439 inhibits both AURKA and AURKB.

(A) ZM447439 inhibits both AURKA and AURKB. Mitotic HeLa cells were obtained by exposure to nocodazole for 16 h followed by mechanical shake off. The cells were incubated with the indicated concentrations of ZM447439 for 2 h. Nocodazole and MG132 were included to prevent mitotic exit. Lysates were prepared and activated phospho-AURKAThr288 and AURKBThr232 were detected with immunoblotting. The asterisk indicates the position of an AURKB-like protein (see text). Uniform loading was confirmed by immunoblotting for actin.

(B) ZM447439 induces mitotic slippage. HeLa cells expressing histone H2B-GFP were transfected with control or AURKB siRNA. The cells were then incubated with 1 µM of ZM447439. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal line represents one cell (n=100). Key: light grey=interphase; black=mitosis (from DNA condensation to anaphase or mitotic slippage); dark grey=interphase after mitotic slippage; truncated bars=cell death.

Figure S4. Alisertib inhibits both AURKA and AURKB in HCT116 cells.

(A) Both AURKA and AURKB are inhibited by Alisertib. Mitotic HCT116 cells were obtained by exposure to nocodazole for 16 h followed by mechanical shake off. The cells were incubated with the indicated concentrations of Alisertib for 2 h. Nocodazole and MG132 were included to prevent mitotic exit. Lysates were prepared and activated phospho-AURKAThr288 and AURKBThr232 were detected with immunoblotting. The asterisk indicates the position of an AURKB-like protein (see text). Uniform loading was confirmed by immunoblotting for actin.

(B) Alisertib induces mitotic slippage in an AURKB-dependent manner. HCT116 cells expressing histone H2B-GFP were transfected with control or AURKB siRNA. The cells were exposed to buffer or the indicated concentrations of Alisertib. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal line represents one cell (n=50). Key: light grey=interphase; black=mitosis (from DNA condensation to anaphase or mitotic slippage); dark grey=interphase after mitotic slippage; truncated bars=cell death.

(C) Alisertib suppresses long-term survival in a concentration-dependent manner. HCT116 cells were seeded on 60-mm culture plates and grown in the presence of 250 nM or 1 µM of Alisertib. After 24 h, the cells were washed gently and propagated in normal medium for another 10-12 days. Colonies were fixed and stained with crystal violet staining solution (examples of the plates are shown). Average±SD from three independent experiments.

Figure S5. Barasertib induces mitotic slippage.

HeLa cells expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of Barasertib. Individual cells were then tracked for 24 h with time-lapse microscopy. The percentage of cells that underwent mitotic slippage during the imaging period was quantified. Mitotic slippage occurred in cells that first formed a metaphase plate (grey) and those that did not (black).

Figure S6. Downregulation of p53 in HepG2 increases sensitivity to Barasertib.

(A) Downregulation of p53 sensitizes cells to Barasertib. HepG2 cells were transfected with either control or p53 siRNAs. The cells were treated with buffer or the indicated concentrations of Barasertib. Individual cells were then tracked for 24 h with time-lapse microscopy (bright field). Each horizontal line represents one cell (n=50). Key: light grey=interphase; black=mitosis (from round up to cleavage furrow ingression or mitotic slippage); dark grey=interphase after mitotic slippage; truncated bars=cell death.

(B) Downregulation of p53 sensitizes cells to Barasertib-mediated mitotic slippage. Live-cell imaging after siRNA transfection and Barasertib treatment was described in panel (A). The percentage of cells that underwent mitotic slippage during the imaging period was quantified.

SUPPLEMENTAL VIDEO LEGENDS

Video S1. Live-cell imaging of control HeLa cells.

HeLa cells expressing histone H2B-GFP were subjected to time-lapse microscopy. Channels for bright field (left) and histone H2B-GFP (right) are shown. The representative video was captured at 5 min/frame.

Video S2. Low concentration of Alisertib delays mitotic exit.

HeLa cells expressing histone H2B-GFP were exposed to 250 nM of Alisertib and subjected to live-cell imaging. Channels for bright field (left) and histone H2B-GFP (right) are shown. The representative video was captured at 5 min/frame.

Video S3. High concentration of Alisertib induces mitotic slippage.

HeLa cells expressing histone H2B-GFP were exposed to 750 nM of Alisertib and subjected to live-cell imaging. Channels for bright field (left) and histone H2B-GFP (right) are shown. The representative video was captured at 5 min/frame.

Video S4. Barasertib induces mitotic slippage without metaphase.

HeLa cells expressing histone H2B-GFP were exposed to 25 nM of Barasertib and subjected to live-cell imaging. Channels for bright field (left) and histone H2B-GFP (right) are shown. The representative video was captured at 5 min/frame.

Video S5. Barasertib induces mitotic slippage after formation of metaphase plate.