1. 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 Cancer

2. Supplementary Fig. 2. Chemical structures of HDIs. SK-7068 LAQ824 Functional moiety Linker Cap MS-275 Butyric acid Valproic acid Apicidin

3. 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) Cell cycle phase (%) Non-transformed cells(IE) MPM2 Fluorescence G1 S G2/M SubG1 0h 12h Cell cycle phase (%) MPM2 Fluorescence G1 S G2/M SubG1 0h 12h

4. 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)

5. 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) b SNU-16 IE

6. 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 Ac-Lys 0 12 24 hrs Aurora-A InputIP: 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.

7. 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) Relative HDAC Activity (%) IP:HDAC6 SK-7068 0 12 24 hrs LAQ824 0 12 24 hrs Relative HDAC Activity (%) IP:HDAC6 a b

8. 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

9. 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

10. 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-7068LAQ-824 Caspase-8 Bid  -tubulin a b ControlHDIHDI+zVAD-fmkHDI+zIETD-fmk c