Supplemental Information

SMK-17, a MEK1/2-specific inhibitor, selectively induces apoptosis in

β-catenin-mutated tumors

Masaki Kiga, Ayako Nakayama, Yuki Shikata, Yukiko Sasazawa, Ryo Murakami,

Nakanishi Toshiyuki, Etsu Tashiro, Masaya Imoto

Supplemental Figure Legends

Figure S1. SMK-17-mediated inhibition of ERK1/2 phosphorylation and induced

expression of cleaved PARP in vivo.

After a single oral dose of SMK-17, tumors were excised from A375- and

SW48-xenograft mice 6 h after dosing, and the tumor lysates were analyzed by western

blot.

Figure S2. SMK-17 upregulated BIM expression.

Cells were treated with the indicated concentrations of SMK-17. After 18 h, cell lysates

were probed for ERK1/2, phospho-ERK1/2 (p-ERK1/2), and BIM via western blot.

Figure S3. Comparative c-Myc protein and mRNA expression levels of the tumor cell

lines.

[A] Exponentially growing A375, HT-29, HCT 116, SW48, and colo-205 cells were

lysed in RIPA buffer and subjected to Western blotting using anti-c-Myc or anti-β-actin

antibody. [B] The intensities of each band were quantified and the expression level of

c-Myc was normalized to β-actin. [C] Total RNA of each tumor cells was extracted

using TRIzol reagent (Invitrogen) and transcribed into cDNA using MM-LV Reverse

Transcriptase (Promega) according to the manufacturer’s instructions. Quantitative PCR

was performed with a Thermal Cycler Dice (TaKaRa), using a SYBR Premix Ex Tag

Kit (TaKaRa). The relative amount of c-Myc mRNA was normalized by Rpl37A

mRNA. The primers designed for quantitative Real-Time RT-PCR analysis were as

follows: c-Myc sense 5’-CCTACCCTCTCAACGACAGC-3’ and antisense

5’-CTCTGACCTTTTGCCAGGAG-3’; Rpl37A sense

5’-ATTGAAATCAGCCAGCACGC-3’ and antisense

5’-GCAGGAACCACAGTGCCAGATCC-3’.

Figure S4. SMK-17 induced apoptosis in APC-mutated cell lines.

Cells were treated with the indicated concentrations of SMK-17. After 18 h, cell lysates

were probed for ERK1/2, phospho-ERK1/2 (p-ERK1/2), c-Myc, BIM, survivin, and

cleaved PARP by western blotting.

Figure S5. Comparative TOPFlash assays of the tumor cell lines.

TCF4 transcriptional activities of cell lines expressing wild-type or mutated β-catenin or

mutated APC were compared. Cells were co-transfected with the TOPFlash and Renilla

luciferase plasmids. Transfected cells were incubated for 48 h, and their luciferase

activity was measured. The ratio of the firefly luciferase intensity of TOPFlash to that

of continuous Renilla luciferase was used as an indicator of TCF4 transcriptional

activity.

Figure S6. Comparative nuclear β-catenin expression among various tumor cell lines.

The expression of nuclear β-catenin among cell lines expressing wild-type or mutated

β-catenin or mutated APC were compared. Cells were fixed with 3% paraformaldehyde

and immunostained with antibodies to β-catenin (anti-β-catenin-alexa-488-conjugated

(Cell Signaling)) and Hoechst33258 and then observed under a confocal laser scanning

microscope system FV1000 (Olympus). The relative amount of β-catenin expression

localized in nucleus was quantified with ImageJ software.

Supplemental Figure S1�

Cleaved PARP

p-ERK1/2

ERK1/2

SMK-17 (mg/kg)

���0 50 200 400

SW48 cells �

SMK-17 (mg/kg)

��0 � 50 200 400

A375 cells �

SMK$17,(a(MEK1/2$specific(inhibitor,(selec:vely(induces(apoptosis(in(β$catenin$mutated(tumors�Masaki&Kiga,&Ayako&Nakayama,&Yuki&Shikata,&Yukiko&Sasazawa,&Ryo&Murakami,&Nakanishi&Toshiyuki,&Etsu&Tashiro,&Masaya&Imoto�

ERK1/2

p-ERK1/2

BIM

β-actin

SMK-17 (μM) 3.0

10

0

Colo-205cells

3.0

10

0

SW48cells

3.0

10

0

HCT 116cells

Supplemental Figure S2�

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Supplemental Figure S3�

SMK-17 (µM)

c-Myc

p-ERK1/2

Survivin

BIM

β-actin

Cleaved PARP

3.0

1.0

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0

SW620 cells

ERK1/2

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SW480 cells

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Supplemental Figure S4�

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Supplemental Figure S6�