Supplementary Fig. 1. (a) Cells were treated with DMSO or with 1 µM of SK-7068 or LAQ824 for 18 hours. They were then fixed with 70% ethanol, stained with propidium iodide, and subjected to flow cytometric analysis. Cell population percentages in each cell cycle phase (G1, S, or G2/M) were calculated with DNA content histograms, and the population in the sub-G1 phase was excluded. The values shown represent the means of three experiments; means, ± SDs. (b) The cells were treated with DMSO or with 1 µM of SK-7068 or LAQ824 for 12 hours, fixed and immunostained with MPM2 antibody. The MPM2 signals were analyzed by flow cytometry. MPM2-positive cells were then counted, and percentages were calculated. MPM2 signal induction fold data was provided at specified time points, divided by the value of the DMSO-treated control. The data shown represent the averages of three experiments; means, ± SD.

a

b

Non-transformed cells

Non-transformed cells

Cancer

Cancer

Supplementary Fig. 2. Chemical structures of HDIs.

SK-7068

LAQ824

Functional moiety

Functional moiety

Linker

Linker

Cap

Cap

MS-275

Butyric acid

Valproic acid

Apicidin

Supplementary Fig. 3. Cancer (SNU-16) and non-transformed (IE; rat intestinal epithelial) cells were treated with 1 µM of MS-275, 1 mM of butyric acid, 1 µM of valproic acid, or 1 µM of apicidin for 18 (upper panel; cell cycle profile) or 12 hrs (lower panel; MPM2 staining). Cell cycle analysis and MPM2 staining were performed as described in “Materials and Methods”. The values shown represents the means of three experiments; means, ± SDs.

Cancer cells (SNU-16)C

ell c

ycle

ph

ase

(%)

Non-transformed cells(IE)

MP

M2

Flu

ore

scen

ce G1 S G2/M SubG1 G1 S G2/M SubG1 G1 S G2/M SubG1 G1 S G2/M SubG1

0h 12h 0h 12h 0h 12h 0h 12h

Cel

l cyc

le p

has

e (%

)M

PM

2 F

luo

resc

ence

G1 S G2/M SubG1 G1 S G2/M SubG1 G1 S G2/M SubG1 G1 S G2/M SubG1

0h 12h 0h 12h 0h 12h 0h 12h

Supplementary Fig. 4. The localizations of -tubulin and mitotic spindles during cell division were analyzed, both in the non-transformed (M13SV1) and cancer (HeLa) cells. Cells were synchronized using a double thymidine block and then treated with DMSO or 1 µM of SK-7068. Cells at telophase were fixed and immunostained, then visualized under microscopy.

Non-transformed (M13SV1)

Supplementary Fig. 5. HDI selectively downregulates the mitotic centrosomal protein Aurora-A in cancer cells. (a) Cancer (SNU-620) or non-transformed (IE) cells were treated with 1 µM of SK-7068 for the indicated times, and the expressions of Aurora-A, Aurora-B, and Survivin were then determined by immunoblotting. (b) SNU-16 cells were treated with HDIs at the indicated concentrations for 12 or 24 hrs, and the expressions of Aurora-A were visualized by immunoblotting.

SNU-620

SK-7068 0 12 24 hrs

Aurora-A

Aurora-B

Survivin

-Tubulin

0 12 24 hrs

a

Aurora-A

-tubulin

0 0.1 0.3 0.6 1 M

SK-7068 (12hrs)

Aurora-A

-tubulin

0 0.1 0.3 0.6 1 M

SK-7068 (24hrs)

Aurora-A

-tubulin

0 0.1 0.3 0.6 1 M

LAQ824 (12hrs)

Aurora-A

-tubulin

0 0.1 0.3 0.6 1 M

LAQ824 (24hrs)

bSNU-16

IE

IP:Hsp90

0 12 24 hrs

Aurora-A

Hsp90

Ac-Lys

SK-7068

Input

Aurora-A

0 12 24 hrs

Hsp90

IP:Hsp90

0 12 24 hrs

Aurora-A

Hsp90

Ac-Lys

LAQ824

Input

Aurora-A

0 12 24 hrs

Hsp90

a

b

0 12 24 hrs

Aurora-A

Hsp90 Hsp90

Ac-Lys

0 12 24 hrs

Aurora-A

Input IP: Hsp90

Non-transformed cells (HaCat)

Supplementary Fig. 6. (a) HDIs induce Hsp90 acetylation. HCT116 cells were treated with 1 µM of LAQ824 or SK-7068 for indicated times. Hsp90 was immunoprecipitated with antibody against Hsp90 and degrees of Hsp90 acetylations were determined using antibody against Ac-Lys. (b) HaCat cells were treated with 1 µM of LAQ824 for indicated times. Hsp90 was immunoprecipitated with antibody against Hsp90 and degrees of Hsp90 acetylations were determined using antibody against Ac-Lys.

Supplementary Fig. 7. (a) Hsp90 and Hsp70 were not co-immunoprecipitated with Aurora-A in non-transformed cells. Cancer (HCT116) and non-transformed (HaCat) cells were treated with 1 µM of LAQ824 and/or MG132 for indicated times, and then subjected to an immunoprecipitation assay. The lysates were immunoprecipitated using Aurora-A antibody, and were then immunoblotted with indicated antibodies. (b) HCT116 cells were treated with 1 µM of LAQ824 or SK-7068 for indicated times and then hole cell lysates were immunoprecipitated with antibody against HDAC6 and collected immune complexes were subjected to HDAC inhibition assays. HDAC activity assays were conducted using a HDAC fluorescent activity assay kit (BIOMOL, Plymouth Meeting, PA, USA), in accordance with the manufacturer’s instructions. Fluorescence was measured using an LS 55 luminescence spectrometer (Perkin-Elmer, Waltham, MA, USA).

LAQ824 - + + + +MG132 - - - + +

0 12 24 12 24

Input

Hsp90

Hsp70

Aurora-A

- + + + +- - - + +

0 12 24 12 24 hrs

IP: Aurora-A

Cancer cells (HCT116)

Hsp90

Hsp70

Aurora-A

LAQ824 - + + + +MG132 - - - + +

0 12 24 12 24

Input

- + + + +- - - + +

0 12 24 12 24 hrs

IP: Aurora-A

Non-transformed cells (HaCat)

0

10

20

30

40

50

60

70

80

90

100

110

Rel

ativ

e H

DA

C A

ctiv

ity

(%) IP:HDAC6

SK-7068 0 12 24 hrs0

10

20

30

40

50

60

70

80

90

100

110

LAQ824 0 12 24 hrs

Rel

ativ

e H

DA

C A

ctiv

ity

(%)

IP:HDAC6

a

b

Supplementary Fig. 8. (a) HCT116 cells were treated with 1 µM of SK-7068 for indicated times and then cell lysates were immunoprecipitated with Aurora-B. Collected immune complexes were subjected to SDS-PAGE and immunoblotted with indicated antibodies. (b) 17-AAG, a Hsp90 inhibitor, induced down-regulation of Aurora-A in HCT116 cells. Cells were treated with 17-AAG (lane 1: 0 mM, lane 2: 1 mM, lane 3: 5 mM, lane 4: 10 mM) for 48 hrs and then immunoblotted with indicated antibodies.

IP:Aurora-B

0 12 24 hrs

a

Aurora-B

Hsp90

Hsp70

HDAC6

0 12 24 hrs

Input

SK-7068

b

Aurora-A

Tubulin

17-AAG

Aurora-B

HCT116

Supplementary Fig. 9. (a) VX-680 causes accumulation of cells at G2/M phase. SNU-620 cells were treated with 300 nM VX-680. DNA content of cells collected at the indicated time points was assessed by flow cytometric analysis of cells labeled with propidium iodide. (b) Protein expressions of Aurora kinases were not altered by VX-680. SNU-620 cells were treated with VX-680 and/or MG132 (300 nM, 1 uM respectively) for indicated times, and then subjected to an immunoprecipitation assay. The lysates were immunoprecipitated using Aurora-A or Aurora-B antibody, and were then immunoblotted with indicated antibodies.

VX-680 - + + + +MG132 - - - + +

0 12 24 12 24

Input

Hsp90

Hsp70

Aurora-A

- + + + +- - - + +

0 12 24 12 24 hrs

IP: Aurora-A

SNU-620

Hsp90

Hsp70

Aurora-B

VX-680 - + + + +MG132 - - - + +

0 12 24 12 24

Input

- + + + +- - - + +

0 12 24 12 24 hrs

IP: Aurora-B

SNU-620

a

0 h 12 h 24 h

b

HDI - - + - + - +

0 6 12 24 hrs

FAS

-actin

FASL

TRAIL

Supplementary Fig. 10. HDI does not affect on the death receptor pathway. (a) HDI does not affect the transcriptional level of FAS, FASL, or TRAIL. SNU-620 cells were treated with DMSO or 1 µM of SK-7068 for the indicated times, and mRNA expressions of FAS, FASL, or TRAIL were then analyzed by RT-PCR. (b) HDIs do not affect Caspase-8/Bid pathway. SNU-620 cells were treated with 1 µM of SK-7068 or LAQ824 for the indicated times and cell extracts were analyzed by immunoblotting with antibodies specific for caspase-8, and bid. (c) HDI-induced apoptosis is not depend on the Caspase-8 activity. SNU-620 cells were treated for 24h with DMSO, 1 µM of SK-7068, 1 µM of SK-7068 plus 50 µM of zVAD-fmk (pan-caspase inhibitor), or 1 µM of SK-7068 plus 50 µM of zIETD-fmk (caspase-8 inhibitor). Cells were analyzed by flow cytometry after propidium iodide staining.

0 12 24 0 12 24 hrs

SK-7068 LAQ-824

Caspase-8

Bid

-tubulin

a

b

Control HDI HDI+zVAD-fmk HDI+zIETD-fmk

c