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Matsushita et al. Hepatoma Res 2018;4:61 I http://dx.doi.org/10.20517/2394-5079.2018.81 Page 3 of 17

by sodium dodecyl sulfate -poly- acrylamide gel electrophoresis on 7.5% or 10%-20% XV PANTERA gels
and transferred onto polyvinylidene fluoride membranes using a tank transfer apparatus. The membranes
were blocked with 0.5% skim milk in PBS overnight at 4 °C. Antigens on the membranes were detected with
enhanced chemiluminescence detection reagents (GE Healthcare UK Ltd., Buckinghamshire, UK). Membranes
were incubated with primary antibodies [Supplementary Table 1] for 1 h at room temperature, followed
by three 10-min washes with 1xPBS/0.01% Tween 20. Membranes were then incubated with commercial
secondary antibodies [Supplementary Table 1], followed by three 15-min washes with 1xPBS/0.01% Tween 20.
[22]
The primary mouse monoclonal antibody against FIR’s C-terminus (6B4) was described previously .
Small molecular chemical compounds screening against FIRΔexon2
Small molecular chemical compounds against His-tagged FIR (His-FIR) and His-tagged FIRΔexon2 were
screened among 2,3275 chemicals of NPDepo at RIKEN as described previously [35-37] . Briefly, His-FIR (645 μg/mL)
and FIRΔexon2 (652 μg/mL) proteins were applied to NPDepo array that contains 2,3275 natural chemical
compounds. FIRΔexon2 inhibitor BK697 was diluted in dimethyl sulfoxide (DMSO) at the concentration of
10 mm, stored in room temperature, treated into HeLa and NUGC4 cell lines with different concentrations
at different time intervals (see details in figure legends). Briefly, on day one, NUGC4 cells or HeLa cells were
prepared in Iscove’s modified Dulbecco’s medium supplemented with 10% FBS. On day two, candidate
chemicals that inhibit FIRΔexon2 protein were diluted in DMSO at the concentration of 10 mm and added
as 10 L or 20 L/well/2 mL in the medium (final concentration in medium was 50 mol/L and 100 mol/L
respectively) or added as 20 L or 60 L/well/2 mL medium (final concentration in medium was 100 mol/L and
300 mol/L respectively). 100 mol/L or 300 mol/L of BK697 was treated to NUGC4 cells for 24 h, 50 mol/L or 100 mol/L
of BK697 was treated to NUGC4 cells or HeLa cells for 6 h, 24 h and 48 h at 37 °C in a CO incubator.
2

Screening procedures of natural small molecular weight chemical compounds that potentially
bind to FIR/FIRΔexon2
Small molecular weight chemical compounds potentially bound to FIRΔexon2 were previously identified
[Figure 1] from the NPDepo at RIKEN, which were a collection of the isolates from natural products, build by
Dr Hiroyuki Osada (RIKEN, Japan) and his coworkers [35-37] .

Procedure of in silico screening
In the process for searching potent compounds, in silico screening was performed from the commercial
chemical database. First, 1000 compounds were selected from the Namiki database that contains 5 million
chemical entries, from the viewpoint of structural similarity to natural product that was identified to be bound
to FIR in our previous work. Second, 125 compounds were extracted from the selected 1000 chemicals in terms
of the electrostatic potential caused by the distribution of positive and negative charges. Finally, 5 compounds
were purchased from a supplier for experimental assay. Namiki database (Namiki Shoji Co., Ltd., Tokyo, Japan,
https://www.namiki-s.co.jp/english/) was a collection of commercially available screening-candidate chemicals.

Display of three-dimensional structure of F-box and WD repeat domain-containing 7 (FBW7)
To examine the possibility of molecular interaction between FBW7 and FIRs from structural viewpoint,
two crystal structures were downloaded from protein data bank (PDB , https://www.rcsb.org/). One is a
complex structure of FBW7 (PDB entry code: 2OVR). The other is the structure of an U2AF homology motif
(UHM) domain in complex with UHM-ligand motif (ULM) of SAP155 (PDB entry code: 2PEH). Both crystal
structures were visualized by PyMOL (DeLano, W. L.; The PyMOL Molecular Graphics System, Schrödinger,
LLC).

MTS assay (Cell proliferation assay)
One day before the chemical treatment, cells were cultured in 100 μL medium in flat-bottomed 96-well plates