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Supplemental Figures

Figure S1. UCH-L1 interacts with overexpressed CDK2.

COS-7 cells were transfected as indicated. Proteins were immunoprecipitated and analyzed by

immunoblotting.

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Figure S2. Neurodegenerative disease-linked mutant SOD1 interacts with CDK4, but did not enhance

CDK4 activity.

(A) Amyotrophic lateral sclerosis-linked mutant A4V SOD1 interacts with CDK4, but WT SOD1 does

not. HeLa cells were transfected with FLAG-tagged SOD1 (WT, A4V) or empty vector (control).

Proteins were immunoprecipitated and analyzed by immunoblotting.

(B) Cell-based kinase assay. COS-7 cells were co-transfected with indicated plasmids.

Phosphorylation of Rb was analyzed by immunoblotting.

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Figure S3. UCH-L1 does not affect the interaction levels between CDKs and cyclins, or CDK

inhibitors.

(A-E) COS-7 cells were transfected with the indicated plasmids. Proteins were immunoprecipitated

and analyzed by immunoblotting.

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Figure S4. Alignment of amino acid residues of CDKs.

For the alignment of CDK1, CDK2, CDK4, CDK5 and CDK6, the positions where at least three

amino acid residues are identical are shown in grey boxes.

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Figure S5. Amino acid residues of UCH-L1 that are involved in the interaction with CDK4

(A) Alanine-scanning mutagenesis of UCH-L1. Lysates of COS-7 cells transfected with the indicated

constructs were immunoprecipitated with anti-FLAG antibody, and analyzed by immunoblotting.

(B) Structural model for human UCH-L1. Arg-63 is shown in light green, Glu-174, Asp-176 and

His-185 are in magenta, and aa 160–190 amino acid residues are shown in yellow. The NCBI

structural model (mmdbId:38174) was used.

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Figure S6. UCH-L1 enhances proliferation of COS-7 cells.

(A and B) COS-7 cells were transfected with UCH-L1 or empty vector (control). At 48 h

post-transfection, relative numbers of viable cells were measured by ATP-based cell viability assays

(A), and cell death by LDH-release assay (B). Mean values are shown with S.E. (n = 4). *P < 0.05;

n.s., not significant.

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Figure S7. Effect of UCH-L1 overexpression on the proliferation of cells treated with CDK4

inhibitors, and in cells where CDK4 is knocked down.

(A) HeLa cells were transfected with the indicated plasmids (Control: empty vector). At 3 h

post-transfection, cells were incubated with 5 μM CDK4 inhibitor for 24 h. Relative numbers of viable

cells were measured. Mean values are shown with SEM (n = 3).

(B) HeLa cells were transfected with CDK4 siRNA, incubated for 72 h, and analyzed by immunoblot.

(C) HeLa cells were transfected with CDK4 siRNA. At 24 h post-transfection, cells were then

transfected with the indicated plasmids, and relative numbers of viable cells measured at 48 h

post-transfection. Mean values are shown with SEM (n = 3).

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Figure S8. Interaction between UCH-L1 and D144N CDK5.

HeLa cells were transfected with the indicated plasmids. Proteins were immunoprecipitated and

analyzed by immunoblotting.

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Figure S9. Effect of UCH-L1 siRNAs on proliferation of HeLa cells.

HeLa cells were transfected with the indicated siRNA (Control: EGFP siRNA for human cells), and

viable cell numbers were counted 48 h after transfection. Mean values are shown with SEM (n = 3).

Although mUCH-L1 siRNA-B enhanced proliferation, this effect is likely an off-target effect because

mUCH-L1 siRNAs were designed to be used in mouse cells.

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Figure S10.

(A) Body weight of mice with xenograft tumors. Mean values are shown with S.E. (n = 5/group). n.s.,

not significant.

(B) Red blood cell and white cell numbers of xenograft tumors. Mean values are shown with S.E. (n =

5/group). n.s., not significant.

(C) Splenocytes were isolated from siRNA-treated nude mice. Isolated splenocytes were seeded into

96-well culture plates with RPMI-1640 containing 10% FBS at a density of 20, 10, 5, or 2.5 105

cells/well (E/T ratio = 20, 10, 5, 2.5, respectively). Subsequently, MCF-7 target cells were added to

each well at a density of 1 105 cells/well. Six hours after the addition of target cells, an amount of

released LDH in culture medium was measured using a CytoTox 96 Non-Radioactive Cytotoxicity

Assay (Promega) according to the manufacturer’s protocol. The percentage of specific lysis as

cell-mediated cytotoxicity was calculated by the following formula: cytotoxicity (%) = (experimental

LDH release – effector cell spontaneous LDH release – target cell spontaneous LDH release) / (target

cell maximum LDH release – target cell spontaneous LDH release) 100.

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Figure S11. Schematic representation of a model for the enhancement of proliferation by UCH-L1.

UCH-L1 interacts with CDK1 and CDK4, and enhances their kinase activity. UCH-L1 enhances cell

proliferation through the enhancement of CDK1 and CDK4 activities.