Martin et al Supplementary Data

Figure S1:

Representative time lapse montages of HeLa cells treated with either A. DMSO (Control), or

B. 1 M Alisertib or C. 1 M MK5108. Frames were captured at intervals of 30 min. The

delay in mitosis and failed cytokinesis is seen with A

Figure S2:

Examples of nuclear phenotypes assessed after Aurora siRNA or inhibitor treatment.

Figure S3:

Asynchronously growing HeLa cells were treated with the indicated doses of drugs,

harvested after 48 h drug treatment and immunoblotted for the indicated markers of Aurora B

(pH3 Ser10), mitosis (pMEK Thr286), and apoptosis (cPARP). The data are the mean and SD

from at least three independent determinations. C. Representative immunoblots quantitated

in A and B. and Figure 2A, B.

Figure S4:

HeLa cells were blocked in mitosis with nocodazole and then MG132 to prevent exit, then

treated with ZM447439 in a dose response for 4 h. Lysates were prepared from each drug

treatment and immunoblotted for pH3 as marker of Aurora B activity, and pAurora A Thr288

(pAURKA) as a marker of Aurora A activity. Cleaved PARP (cPARP) was assessed from

asynchronously growing HeLa cells treated with the same drug concentration for 72 h. The

band densities were measured from three experiments and are presented and mean and

standard deviation of the percentage of the untreated control.

Figure S5:

A. The surviving fraction of cells at 1 and 10 M drug from the dose response experiments

shown in Table 2. The data are the mean and SD from at least 4 replicates.

B. HeLa and C33A cells treated with the indicated concentrations of Alisertib or AMG900, or

DMSO alone as a control, were fixed 24 h after drug treatment. The fixed cells were stained

for DNA and microtubules and the proportion of normal, failed mitosis (i.e. cells with either

micronuclei, bi- or multiple nuclei), and apoptotic cells was assessed in >200 cells for each

condition.

Figure S6:

HeLa cells were treated with 0.3 and 1 M Alisertib or AMG900, and 1 M MK5108

(AURKAi), 1 M AZD2811 (AURKBi), and in combination. Cells were followed for 72 h

after drug treatment and the time to and in first, second and third mitosis, and to apoptosis or

where apoptosis was during mitosis was assessed for >70 cells for each condition. This data

is representative of three separate experiments.

Figure S7:

HeLa xenograft tumour excised 6 h after final Alisertib treatment were H&E stained and

immunostained for phospho-H3 Ser10 (pH3).

Figure S8:

HeLa cells were treated for 24 h with the indicated concentration of drug then fixed and

immunostained for phospho-H3 Ser10 (pH3) and DNA. Bar = 10 M.

Figure S9:

HeLa cells were treated with the indicated concentrations of drugs for 24 h then fixed and

immunostained for DNA, Aurora B, and -tubulin for microtubules (MT). Bar = 10 M.

Figure S10:

HeLa xenograft tumours, either untreated (A) or harvested 24 h after the final Alisertib

treatment (B, C) were stained for DNA, Aurora B and -tubulin to show the microtubules

(MT). Midbodies positive for Aurora B and microtubule staining are indicated by the open

arrowheads, and defective mitosis with the filled arrowheads. C shows a magnified view.

 

Supplementary Table 1: IC50 data for Aurora A and B inhibition in mitotically arrested

HeLa (nM).

Supplementary Table 2: IC50 data for cell lines. IC50 values were determined from 7 point

dilution series for each drug. Each cell line was analysed in technical replicates (4) and each

determination was performed twice. IC50 values were calculated using R studio.

pH3  pAurA  cPARP Alisertib  350  29  110 AMG900  8  14  8 

ZM447439 165  670  >3000 

   IC50 (nM) Cell Line  Alisertib AMG900 MK5108  AZD2811HPV‐ve         C33A  350  2,200  2,200  6,000 SCC25  3,000  1,000  2,400  >10000          HPV+ve         CaSki  133 

Martin et al. Supp Figure S1

1 M Alisertib B

A Control

1 M MK5108 C

Martin et al., Supp Figure S2

DN

A M

t+D

NA

Normal Micronuclei Binuclear Multinuclear

0

2000

4000

6000

8000 Alisertib pH3

%C

ontro

l

0

500

1000

1500

2000Alisertib pMEK

%C

ontro

l

0 10 100 1000

0 10 100 1000 0

100

200

300

400

500 AMG900 pH3

%C

ontro

l

0

200

400

600

800

1000 AMG900 pMEK

%C

ontro

l

0 10 100 1000

0 10 100 1000

BA

Martin et al. Supp Figure S3

0

50

Alisertib cPARP

MLN8237 (nM)0 10 100 1000

0

50

100

AMG900 (nM)0 10 100 1000

AMG900 cPARP

pAURKAAURKA

-tubulin

pH3 Ser10AURKB

-tubulincPARP

0 10 30 100 300 1000 3000 AMG900 (nM)

C

Martin et al. Supp Figure S4

0

50

100

150

ZM447439 (nM)

0

50

100

150

ZM447439 (nM)

0 10 100 1000

0 10 100 1000

ZM447439 pH3

ZM447439 pAURKA

%C

ontr

ol%

Con

trol

0

50

100

%C

ontr

ol

ZM447439 (nM)0 10 100 1000

ZM447439 cPARP

Martin et al. Supp Figure S5

M drug0

1020304050607080

1 10 1 10 1 10 1 10

Aliser b AMG900 MK5108 AZD2811

Surviving Frac on

C33A SCC25 Caski HeLa VK2 Ect1HPV nega ve HPV posi ve

NormalFailedApoptosis

HeLa (HPV+ve) C33A (HPV-ve)

% N

ucle

r phe

noty

pe

0%

20%

40%

60%

80%

100%B

% S

urvi

val

A

0 10 20 30 40 50 60 701

4

7

10

13

16

19

22

25

28

31

34

37

40

43

46

49

52

55

58

61

64

67

70

1 M Aliser b

# to M1# in M1

# to M2# in M2

# to M3# in M3

# to apop

mit apop

Time a er treatment (h)0 10 20 30 40 50 60 70

1

4

7

10

13

16

19

22

25

28

31

34

37

40

43

46

49

52

55

58

61

64

67

70

1M AMG900

Time a er treatment (h)

0 10 20 30 40 50 6013579

1113151719

Control

Time a er treatment (h)

0 10 20 30 40 50 60147

1013

16192225283134

37404346495255

5861646770

AURKAi

Time a er treatment (h)0 10 20 30 40 50 60 70

1

4

7

10

13

16

19

22

25

28

31

34

37

40

43

46

49

52

55

58

61

64

67

70AURKBi

Time a er treatment (h)0 10 20 30 40 50 60 70

147

101316192225283134374043464952555861646770

AURKAi+AURKBi

Time a er treatment (h)

Martin et al., Supplementary Figure S6

0.3 M Alser b

Time a er treatment (h)0 10 20 30 40 50 60 70

147

101316192225283134374043464952555861646770737679828588919497

100

Control Alisertib

H&E

pH3

Martin et al. Supp Figure S7

Martin et al. Supp Figure S8

pH3 DNA

Control

1 M MK5018

1 M Alisertib

0.6 M Alisertib

0.3 M Alisertib

Control

1M MK5018

1M Alisertib

1M AZD

DNA Aurora B MT Combined

Martin et al. Supp Figure S9

A

B

C

Martin et al., Supplementary Figure S10

DNA Aurora B MT Combined