10-4-4 was dissolved in dimethyl sulfoxide (DMSO, Sigma), and stored as a stock solution at 1 mg/mL at -20˚C. It was diluted in cell culture medium or other buffer at indicated concentration when treated. The reagents used in the experiment were described as follows: p38 MAPK inhibitor (SB203580), PKC inhibitor (Go 6983), endocyosis inhibitor (Pitstop 2) were purchased from MedChemExpress.
Cell culture and Transient Transfection
In this study, human HCC cell lines HepG2, Huh-7, Hep3B and human liver cell line LO2 were purchased from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). HepG2, Huh-7 and LO2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM); NCI-H460 were cultured in 1640 medium; Hep3B were cultured in MEM medium, supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotics (100 U/ml of penicillin and streptomycin, Omega Scientific) at 37˚C in a humidified atmosphere containing 5% CO2. Transient transfection was performed using Lipofectamine 2000 (Invitrogen) or RNAiMax transfection reagent (Invitrogen) according to the manufacturer’s instructions.
pCMV-myc-Nur77, pGFP-Nur77AB, pGFP-Nur77ΔAF2, pGFP-Nur77ΔAB, pGFP-Nur77ΔDBD, pGFP-Nur77ΔAB/ΔAF2, pGFP-Nur77LBD, pGFP-Nur77DC3, pGFP-Nur77N150, pGFP-Nur77 were described previously or were constructed using PCR.
siRNA and transfections
The human Nur77-SiRNA, human NaK α3 subunit-SiRNA and control siRNA used were from Sigma. Transfections were performed with RNAiMax transfection reagent (Invitrogen), according to the manufacturer’s recommendations. Two days after transfection, the cells were treated with 10-4-4 for 3 hr (for protein expression analysis) or 12 hr (for apoptosis analysis), then harvested.
Cell proliferation assay
Cells were seeded at 3,000 cells per well in 96-well plate. After 24 hr of incubation, they were treated with 10-4-4 containing 10% FBS for 3 days. The control cells received vehicle containing equivalent DMSO. Viable cells were determined by the addition of MTT ([3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]–phenomenon methosulfate, Sigma) solution to each well, and a sequential incubation for 4 hr at 37°C in the dark. Absorbance value was measured at 550nm by a Bio-Rad 550 micro-plate reader. The results were represented as IC50 (50% inhibiting concentration).
Flow Cytometry assay
Cells were collected by centrifugation at 1,000 rpm for 5 min, then resuspended cells with PBS and incubated with PI, Annixn V, and Flou-3AM for 1 hr and analyzed by flow cytometry (FCM), using the FACScater-plus flow cytometer (Becton Dickinson) to test its cell cycle, apoptosis and calcium.
After extraction, proteins in cell lysates were separated using SDS-PAGE. Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes. 5% non-fat milk was used to blocking nonspecific sites of membranes, then membranes were incubated with PARP, phospho-chk1, phospho-chk2, phospho-cdc2, Cyclin B1, Nur77, phospho-PKC, phospho-Akt, phosph-JNK, phospho-p38, Myc, NaK, GFP and β-actin antibodies: Rabbit monoclonal anti-PARP (Cell Signaling Technology, Cat# 9542, 1:1000 dilution), Rabbit monoclonal anti- phospho-chk1 (Cell Signaling Technology, Cat# 12302, 1:1000 dilution), Rabbit monoclonal anti- phospho-chk2 (Cell Signaling Technology, Cat# 2197, 1:1000 dilution), Rabbit monoclonal anti-phospho-cdc2 (Cell Signaling Technology, Cat# 4539, 1:1000 dilution), Rabbit monoclonal anti-Cyclin B1 (Cell Signaling Technology, Cat# 12231, 1:1000 dilution), Rabbit monoclonal Nur77 (Cell Signaling Technology, Cat# 3960, 1:1000 dilution), Rabbit monoclonal phospho-PKC (Cell Signaling Technology, Cat# 38938, 1:1000 dilution), Rabbit monoclonal phospho-Akt (Cell Signaling Technology, Cat# 13038, 1:1000 dilution), Rabbit monoclonal phosph-JNK (Cell Signaling Technology, Cat# 9251, 1:1000 dilution), Rabbit monoclonal phospho-p38 (Cell Signaling Technology, Cat# 9215, 1:1000 dilution), Rabbit monoclonal phospho-p38 (Cell Signaling Technology, Cat# 9215, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology (Massachusetts, USA). Mouse monoclonal anti-Myc (Santa Cruz Biotechnology, Cat# sc-40, 1:2000 dilution), Mouse monoclonal anti- GFP (Santa Cruz Biotechnology, Cat# sc-9996, 1:2000 dilution) antibodis were purchased from Santa Cruz Biochemical (Santa Cruz, CA). Mouse monoclonal anti-β-actin (Sigma, Cat# A5441, 1:10000 dilution) antibody was purchased from Sigma (St. Louis, MO). Na+/K+-ATPase α3 (ab2826, 1:1000) antibody was purchased from abcam (Abcam Inc. Cambridge, UK). Subsequently, the membranes were incubated with a HRP conjugated secondary antibody (Protein Tech Group, Chicago, IL) at room temperature for 1 hr. HRP-conjugated Goat Anti-Mouse IgG (H+L) (Protein Tech Group, Cat# SA00001-1, 1:10,000 dilution), HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Protein Tech Group, Cat# SA00001-2, 1:10,000 dilution). The signals were observed by an enhanced chemiluminescence system (GE Healthcare; Munich, Germany).
Cells were re-suspended in buffer A [10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM Dithiothreitol (DTT)], centrifuge the cell suspension at 6,000Ⅹg for 30 sec to collect cytoplasmic fraction. Re-suspend pellets in Buffer B [20 mM HEPES-KOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM DTT]. Centrifuge the supernatant at 12,000Ⅹg at 4°C for 15 min.
Co-immunoprecipitation (Co-IP) assays [15, 16, 23, 62]
The protein concentration in cell lysates was diluted to about 1 μg/μL, appropriate antibody was added into 500 μL total protein lysate, incubate for 2 hr. 20 μL protein A-Sepharose beads were added to capture the antigen-antibody complex, then detection of interacting proteins by western blotting.
Immunofluorescence microscopy 
Cells mounted on coverslips were fixed with 4% paraformaldehyde for 10 min; incubated the coverslips for 15 min with 0.1% Triton X-100 to permeabilize the cells. Incubated cell with 1% bovine serum albumin for 30 min at room temperature. Incubated cells with diluted primary antibodies in a humidified chamber for 1 hr and detected with fluorophore conjugated secondary antibodies. The images were taken under an LSM-510 confocal laser scanning microscope system (Carl Zeiss, Oberkochen, Germany).
Na+/K+-ATPase -ATPase activity assay
After treating cells with compounds, assayed NaK -ATPase activity of cell lysis with NaK activity Kit from Beyotime Institute of Biotechnology in China.
Zebrafish husbandry, Chemical treatments  and Photography and image analysis 
Transgenic line, Tg (fabp10:rtTA2s-M2; TRE2: EGFP-krasV12) (gz32Tg) in a Tet-On system to control the hepatocyte-specific expression of oncogenic krasV12 , was used in this study.
Doxycycline (Dox) (Sigma, D9891) was added at dose of 75 μg/mL from 3 to 7dpf to induce kras expression. The larvae were exposed to 5 μM 10-4-4 at 4 to7 dpf. 20 larvae were randomly selected from each group for imaging. The larvae were anesthetized in 0.08% tricaine (Sigma, E10521) and immobilized in 3% methylcellulose (Sigma, M0521). Each larva was photographed with Olympus microscope (DP72). ImageJ was used to 2D measure the liver size.
Data were expressed as mean±SD. Each assay was repeated in triplicate in three independent experiments. Statistical significance of differences between groups was analyzed by using Student’s t test or ANOVA analysis. p<0.05 was considered significant.