1. Supplementary Figure S1 Supplementary Figure S1. Regulation of localization or stability of Cdc25C by ASK1. Effect of ASK1 expression on the stability and Ser-216 phosphorylation of Cdc25C. HEK 293 cells were transfected with different amounts of ASK1 construct for 48 h, subsequently exposed to cycloheximide (40 μg/ml) for indicated times with serum-free media. Cell lysates were prepared and subjected to SDS-PAGE. Immunoblotting was performed with anti-Cdc25C, anti-p-Cdc25C (Ser-216), anti-FLAG. An anti-GAPDH antibody was used as a loading control for immunoblotting. 0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2 ASK1 (μg) Cdc25C lysate GAPDH p-Cdc25C (Ser-216) FLAG (ASK1) cycloheximide (h) 0 4 8 40 (kDa) 170 130 55 70

2. Supplementary Figure S2. Cdc25C hyperphosphorylation by ND treatment in HEK 293 cells. (a) Effect of ND on Ser-216 phosphorylation and hyperphosphorylation of Cdc25C was determined. HEK 293 cells were treated with ND (200 ng/ml) for the indicated times, and the lysates were assessed by immunoblotting analysis with anti-Cdc25C or anti-p-Cdc25C (Ser-216) antibody. An anti-GAPDH antibody was used as a loading control for immunoblotting. (b) Dephosphorylation of hyperphosphorylated Cdc25C was confirmed. Total lysates from HEK 293 untreated or treated with nocodazole were incubated in the absence or presence of CIP. The protein samples were subjected to SDS-PAGE and then immunoblotted with an anti-Cdc25C antibody and anti-GAPDH antibody. (c) Changes of p-Ser-216 and hyperphospho-Cdc25C after removal of ND were determined. HEK 293 cells were exposed to ND (200 ng/ml) for 16 h, subsequently released into fresh media and harvested at the indicated timepoint. Cell lysates containing equal amounts of protein were separated by SDS- PAGE and immunoblotted with anti-Cdc25C, anti-p-Cdc25C (Ser-216), or anti-GAPDH antibody. Protein levels were analyzed by immunoblotting and quantified by scanning the immunoblots followed by analysis with LabWorks software. The far left lane (c) shows data obtained from untreated asynchronous cells. a GAPDH ND HEK 293 Fold: 1.0 0.7 0.6 0.3 0.2 p-Cdc25C (Ser-216) 0 2 4 8 16 (h) Fold: 1.0 0.8 0.8 0.5 0.3 Cdc25C 0.1 0.2 0.3 40 70 55 (kDa) Hyper-P Hypo-P Hyper-P Hypo-P lysate c 0.2 0.1 0.1 0.1 Release cdc25C p-cdc25C (Ser216) HEK 293 Fold: 1.0 0.3 0.4 0.5 0.4 0.6 C 0 2 4 8 16 (h) Fold: 1.0 0.1 0.2 0.2 0.3 1.0 GAPDH 40 70 55 (kDa) Hyper-P Hypo-P Hyper-P Hypo-P lysate Supplementary Figure S2 b GAPDH ND CIP HEK 293 Cdc25C 40 70 55 (kDa) Hyper-P Hypo-P lysate ---- +-+- -+-+ ++++

3. Supplementary Figure S3. Hyperphosphorylation of Cdc25C in ASK1 -/- cells. ASK1 WT and ASK1 -/- MEF cells were treated with ND for 16 h. Total cell lysates were prepared and subjected to immunoblotting using anti-Cdc25C antibody. An anti-GAPDH antibody was used as a loading control for immunoblotting. Cdc25C 55 Hyper-P Hypo-P ND - + - + 40 (kDa) GAPDH lysate ASK1 WT ASK1 -/- Supplementary Figure S3

4. Supplementary Figure S4. Distribution of cells by double thymidine block and release to normal cell cycle. HEK 293-A1 cells were synchronized at S phase by double thymidine block method. After synchronization, cells were released to normal cell cycle by replacing with serum-containing medium for indicated times. Total cells were collected and subjected to FACS analysis. The data represented as data which X axis represents the quantities for DNA contents and Y axis represents the number of cells. The far left panel (c) shows data obtained from unexposed asynchronous cells. Release C 0 4 6 8 10 (h) Supplementary Figure S4

5. Supplementary Figure S5. Subcellular localization of Cdc25C by ND treatment. HEK 293 cells were treated with ND (200 ng/ml) for 16 h and cells were collected. Cytosolic (C) and nuclear (N) proteins were fractionated as described in Materials and methods. Protein samples were subjected to immunoblotting using anti-Cdc25C, anti-ASK1, anti-lamin B, and anti-tubulin antibodies. C N Lamin B1 tubulin N ND Cdc25C ASK1 70 55 170 130 70 55 (kDa) Hyper-P Hypo-P Supplementary Figure S5

6. Fold :1.0 0.3 0.3 0.4 0.0 0.0 0.0 Hyper-P Hypo-P Hyper-P Hypo-P Hyper-P Hypo-P Fold :1.0 0.4 0.5 0.5 0.2 0.2 0.2 Hyper-P Hypo-P Supplementary Figure S6. Binding of ASK1 to hyperphosphorylated Cdc25C induced by several mitosis inducers. After HEK 293 cells were treated with synchronizing reagents (ND, PTX, and dolastatin 15) for 16 h, cells were lysed and immunoprecipitated with anti-ASK1 (F- 9) agarose. Immunoprecipitates were subjected to immunoblotting analysis with anti-Cdc25C or anti-ASK1 antibody. The expression levels of Cdc25C and ASK1 were determined by immunoblotting with anti-Cdc25C and anti-ASK1 antibodies, respectively. Protein levels were analyzed by immunoblotting and quantified by scanning the immunoblots followed by analysis with LabWorks software. Hyper-P, Hyperphosphorylated Cdc25C; Hypo-P, Hypophosphorylated Cdc25C. IP: ASK1 lysate ASK1 Cdc25C ASK1 Cdc25C 70 55 70 55 170 130 170 130 (kDa) N ND PTX Dolastatin 15 Supplementary Figure S6

7. Supplementary Figure S7. p53-dependent processing of caspase-3 and PARP by ND treatment. Effect of ND on apoptosis in p53 -/- cells was determined. After HCT 116 WT and p53 -/- cells were treated with ND for indicted times, cells were lysed and immunoblotting was performed with anti-cleaved PARP or anti-cleaved caspase-3 antibodies. An anti-GAPDH antibody was used as a loading control for immunoblotting. The expression profiles of cleaved PARP and cleaved caspase-3 in HCT 116 WT and p53 -/- cells were compared by scanning the immunoblots followed by analysis with LabWorks software. HCT116 WT HCT116 p53 -/- cleaved PARP cleaved caspase-3 ND (200 ng/ml) - 16 24 - 16 24 (h) GAPDH p53 Fold: 0.0 1.0 22.0 0.0 1.0 9.7 Fold: 0.0 1.0 11.5 0.0 0.5 2.5 40 15 100 (kDa) 55 lysate Supplementary Figure S7