Shinsuke Fujii
1,3
, Shinji Matsumoto
1
, Satoshi Nojima
2
, Eiichi Morii
2
, and Akira Kikuchi
1,*
1 Departments of Molecular Biology and Biochemistry, 2 Pathology, Graduate School of
Medicine,
3
Interdisciplinary Program for Biomedical Sciences (IPBS), Institute for Academic
Initiatives, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan.
*
Correspondence author. Department of Molecular Biology and Biochemistry, Graduate School of
Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan.
Phone, 81-6-6879-3410.Fax, 81-6-6879-3419.
E-mail:,akikuchi@molbiobc.med.osaka-u.ac.jp.
1

Supplementary Figure S1. Arl4c expression in colorectal and lung cancer.
Colorectal ( A ) and lung ( B ) cancer specimens were stained with anti-Arl4c antibody and hematoxylin. Black boxes show enlarged images. Areas staining positive for Arl4c were classified as follows. Low, 5-20%; middle, 20-50%; high, 50-95%. Scale bars, 200 μm.
Supplementary Figure S2. Arl4c expression in colorectal and lung cancer cell lines.
( A ) Arl4c mRNA levels in uterus cancer cells (HeLaS3), colorectal cancer cells (HCT116 and
DLD-1), and lung adenocarcinoma cells (A549 and NCI-H358) were measured by quantitative
RT-PCR. Relative levels of Arl4c mRNA expression were normalized by GAPDH and expressed as fold-changes compared with expression in HeLaS3 cells. ( B ) Levels of Arl4c mRNA in HCT116 (left panel) and A549 (right panel) cells transfected with β-catenin siRNA or treated with 10 
M U0126 or 10

M IWP2 for 24 hours were measured and expressed as fold-changes compared with levels in control cells. Results are shown as means ± s.d. of three independent experiments.
＊ , P < 0.01.
Supplementary Figure S3 . Arl4c expression is involved in migration and invasion of cancer cells.
( A and B ) HCT116 ( A ) and A549 ( B ) cells were transfected with control or two independent Arl4c siRNAs, and Arl4c mRNA levels were measured by quantitative RT-PCR. Relative Arl4c mRNA levels were normalized by GAPDH and expressed as fold-changes compared with levels in control siRNA transfected cells. Lysates were probed with anti-Arl4c and anti-HSP90 antibodies. ( C and D )
HCT116 cells (5 x 10 5 cells) and A549 cells (2.5 x 10 4 cells) were transfected with control or two independent Arl4c siRNAs and subjected to migration ( C ) and invasion ( D ) assays for 8 hours and
24 hours, respectively. Migration and invasion activities are expressed as percentages compared to controls cells. Scale bars, 200 μm. ( E and F ) HCT116 ( E ) and A549 ( F ) cells stably expressing GFP or Arl4c-GFP were transfected with control or Arl4c #1 siRNA. Lysates were probed with anti-Arl4c and anti-HSP90 antibodies. Closed triangles in ( E ) indicate degradation products of exogenously
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expressed Arl4c-GFP. Arl4c mRNA levels were measured by quantitative RT-PCR using primers encoding the 3’-UTR. Relative
Arl4c mRNA expression levels were normalized by GAPDH and expressed as fold-changes compared with levels in GFP expressing control HCT116 cells. Results are shown as means ± s.d. of three independent experiments.
＊ , P < 0.01; n.s., not significant.
Supplementary Figure S4 . YAP/TAZ function downstream of Arl4c.
HCT116 ( A and C ) and A549 ( B and D ) cells expressing control or Arl4c #1 shRNA, which had been transfected with GFP or FLAG-YAP
5SA
, were cultured for 5 days ( A and B ) or 4 hours ( C and D ) in
3D Matrigel. Then, the cells were stained with phalloidin and DRAQ5, and areas of spheres were calculated (n = 30) ( A and B ). Scale bars, 50 μm. mRNA levels of CTGF and ANKRD1 in the cells were measured by quantitative RT-PCR and expressed as fold-changes compared with GFP trransfected control cells ( C and D ). Lysates were probed with anti-FLAG, anti-Arl4c and anti-clathrin antibodies. Results are shown as means ± s.d. of three independent experiments.
＊ ,
P <
0.01; n.s., not significant.
Supplementary Figure S5 . YAP/TAZ expression in human colorectal and lung cancer.
Colorectal (6 cases) ( A ) and lung (7 cases) ( B ) cancer specimens were stained with anti-YAP/TAZ antibody and hematoxylin. Black boxes show enlarged images. Cells with nuclear YAP/TAZ are expressed as the percentage of YAP/TAZ-positive cells compared with total hematoxylin stained cells in Arl4c-positive or -negative lesions (n = 3,763 cells in colorectal cancer, n = 5,146 cells in lung cancer). Scale bars, 200 μm.
Supplementary Figure S6 . Inhibition of xenogtaft tumor proliferation by in vivo treatment with Arl4c siRNA.
( A ) Control or Arl4c #1 siRNA was injected around the base of developed tumors at 9 days after implantation of HCT116 cells (1 x 10
6
cells) (n = 2). At 14 days of siRNA injection, the nude mice
3

were sacrificed. Dashed lines show the outline of xenograft tumors and arrowheads indicate the positions of xenograft tumors (top pictures), and extirpated xenograft tumors (bottom pictures) are shown. The tumor volumes and weights were measured, and results are shown as means of two experiments (right two graphs). Scale bars, 1 cm. ( B ) Quantitative RT-PCR for Arl4c mRNA expression in xenograft tumors was performed. Relative Arl4c mRNA levels were normalized by
GAPDH and expressed as fold-changes compared with levels in control siRNA injected tumors.
4