Supplemental Information

Supplemental Materials and methods

Patient samples

Samples of tumor and corresponding normal mucosa tissues were from 861 consecutive patients who were recruited between June 2008 and October 2012 in Zhongshan Hospital of Fudan University (Shanghai, China). Clinicopathologic data were retrieved from a prospectively constructed database. Prospective data was collected and sorted by an independent full-time research assistant. All patients included in this study received radical resections of primary tumors according to their tumor sites. The pathological tumor stage was defined according to the seventh edition of the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) TNM classification. For patients without synchronous distant metastases, postoperative adjuvant chemotherapy FOLFOX was given according to the Chinese and NCCN colorectal guidelines. For patients with unresectable distant metastases, chemotherapy FOLFOX or FOLFIRI was given as treatment. For patients with resectable synchronous distant metastases, radical resections were also conducted on the metastases. No anti-EGFR, anti-VEGF or other targeted agents were used. This study was approved by the ethics committee of Zhongshan Hospital, Fudan University. Written informed consent was obtained from each patient in accordance with the Helsinki Declaration.

Plasmid, vector, and infection

To create the ICT1 shRNA-silenced sub cell line, we used the following shRNA sequences designed against the ICT1 gene (NM_001545): 5′-GCTGTTAATGCTTGTCTATAACTCGAGTTATAGACAAGCATTAACAGCTTTTTT-3′ (S1) and 5′-GCAGAATGTGAACAAAGTGAACTCGAGTTCACTTTGTTCACATTCTGCTTTTTT-3′ (S2). The control shRNA sequence was 5′-GATCCTTCTCCGAACGTGTCACGTCTCGAGACGACGCACTGGCGGAGAATTTTTG-3′. Lentiviruses were generated by triple transfection of 80% confluent 293T cells with modified pFH-KD plasmid (Hollybio, Shanghai, China) and helper plasmids pVSVG-I and pCMVΔR8.92 and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. HCT116 and SW1116 cells were transduced with the constructed lentiviruses containing nonsilencing shRNA (negative control, NC) and ICT1 shRNA (ICT1 -S1/S2) and the infection efficiency was observed after 96 h through a fluorescence microscope (Epoch, BioTek, Winooski, VT, USA) for green fluorescent protein (GFP) expression.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and synthesized into complementary DNA (cDNA) by M-MLV Reverse Transcriptase (Promega, Beijing, People’s Republic of China) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction was performed on a Bio-Rad Connect Real-Time PCR platform (Bio-Rad Laboratories Inc, Hercules, CA, USA) using a GXD kit iqSYBR Green (Bio-Rad). In a typical procedure, each PCR reaction mixture, containing 10 µL of 2× SYBR® Premix Ex Taq, 0.8µL of sense and antisense primers (2.5µM), 5µL of cDNA, and 4.2µL of double-distilled water, was run with the following thermal profile: initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 5 s and extension at 60°C for 20 s. The internal control used was β-actin. Relative gene expression levels were calculated using 2-ΔΔCT analysis. The primers used were as follows:

ICT1 (forward): 5′-CAGCCTGGACAAGCTCTACC-3′

ICT1 (reverse): 5′-CTGGAACCTGACTTCTGCCTTG-3′

β-actin (forward): 5′-GTGGACATCCGCAAAGAC-3′

β-actin (reverse): 5′-AAAGGGTGTAACGCAACTA-3′

Western blot analysis

Cells were lysed in 2× sodium dodecyl sulfate (SDS) sample buffer (100mM Tris-HCl [pH6.8], 10mM EDTA, 4% SDS, and 10% glycine). The protein content was measured using the Lowry method. To detect target proteins, equal amounts of protein samples (30µg) were separated by 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with Tris-buffered saline and Tween 20® (TBST; 25mM Tris, pH7.4, 150mM NaCl, and 0.1% Tween 20®) containing 5% nonfat dry milk at room temperature for 1 h. After washing three times with TBST, the membranes were probed with the primary antibody (an anti-ICT1 rabbit monoclonal antibody, an anti-AMPK rabbit polyclonal antibody, an anti-AMPK (Phospho-Thr183/Thr172) rabbit polyclonal antibody, an anti-SAPK/JNK rabbit polyclonal antibody, an anti- SAPK/JNK (Phospho-Thr183/Tyr185) rabbit monoclonal antibody, an anti-p38 rabbit polyclonal antibody, an anti-p38 (Phospho-Thr180/Tyr182) rabbit monoclonal antibody, an anti-Cleaved Caspase-3 rabbit polyclonal antibody, an anti-PARP rabbit polyclonal antibody, or an anti-GAPDH rabbit polyclonal antibody) overnight at 4°C followed by incubation with horseradish peroxidase (HRP)-linked goat anti-rabbit immunoglobulin (Ig) G secondary antibody for 2 h at room temperature. The blots were detected with Amersham ECL Plus Western Blotting Detection Reagents according to the manufacturer’s instructions. GAPDH was used as the reference control.

Cell viability assay

After lentivirus infection, SW1116, HCT116 and HUVEC cells were seeded at a density of 2×103cells/well in in 96-well plates (100µL per well) and were incubated for 1 and 5 days, respectively. Afterward, 20 µL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; 5.0mg/mL) was added into each well and incubated with the cells for 4 h. Then, 100µL of acidic isopropanol (10% SDS, 5% isopropanol, and 0.01M HCl) was added to each well and incubated at 37°C overnight. The absorbance was then measured using a microplate reader (Varioskan™ LUX multimode microplate reader, Thermo Scientific, CA, USA) at 595nm.

Colony formation assay

After lentivirus infection, SW1116 and HCT116 cells were seeded in a volume of 2mL at a density of 500cells/well in six-well plates. The medium was replaced every three days. After 9 days of culture, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. The fixed cells were stained with freshly prepared crystal violet for 20 min. Colony formation was observed through a light/fluorescence microscope and the colonies were counted.

Cell cycle analysis

The cell-cycle distribution was analyzed by FCM. After lentivirus infection, SW1116 cells were seeded in a volume of 5mL at a density of 2×105cells/well in 6cm dishes. Cells were harvested after 40 h of culture, and fixed in 70% ice-cold ethanol overnight at 4°C. After washing thrice with PBS, the cells were stained to determine DNA content using 300µL PBS containing 50µg/mL potassium iodide (Sigma) and 100µg/mL DNase-free RNase (Sigma). The suspension was incubated in the dark at room temperature for 30 min and then subjected to FCM using a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using the ModFit LT DNA analysis program (Version 4 for Modfit LT, Verity Software House, Topsham, ME, USA).

Cell apoptosis analysis

The cell apoptosis was analyzed by FCM. Following lentivirus infection, HCT116, SW1116 and HUVEC cells were seeded in 6 cm dishes at a density of 8×105 cells/dish and incubated for 72 h. Subsequently, the occurrence of apoptosis was detected by Annexin V-APC/7-AAD apoptosis kit (KeyGEN Biotech, Nanjing, China) following the manufacturer’s instructions. The FACSCalibur™ flow cytometer was used to analyze the percentage of cell apoptosis.

Transwell migration assay

The cell migration was examined using the Transwell chambers (8.0-µm pores, 24 wells, Corning Costar, New York, USA). Briefly, SW1116 and HUVEC cells were seeded in upper chambers at a density of 1×105 cells/well in 200 μL DMEM without FBS after 96 h of lentivirus infection. The upper chambers were filled with DMEM containing 10% FBS. After incubation for 24 h at 37°C, the non-migrated cells on the upper surface of the filter were gently removed using cotton swabs and the migrated cells on the lower surface were fixed with 4% paraformaldehyde, stained with crystal violet, and counted (five random fields per well). In addition, the cells that migrated to the lower surface were dissociated and quantified at 570 nm using the Epoch Microplate Spectrophotometer (Biotek).

Signaling Array assay

The array analysis was performed as described above using PathScan Intracellular Signaling Array kit (Cell Signaling Technology, catalog number 7323). Cell lysates were prepared and transferred onto the intracellular signaling array membrane overnight.

The following day, the antibody membrane was incubated with 1× HRP-linked streptavidin and visualized with LumiGLO and peroxide. Images were taken using Bio-Rad gel documentation system.

Statistical analysis

Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using Student’s t-test. A P value of less than 0.05 was considered statistically significant.

Supplemental Figure Legends

FIG. S1 Representative ICT1 depletion images of colony formation in SW1116 and HCT116 cells.

FIG. S2 Representative ICT1 depletion images of cell cycle distribution in SW1116 cells.

FIG. S3 Representative ICT1 depletion images of Annexin V/7ADD staining results in SW1116 and HCT116 cells.

FIG. S4 Representative ICT1 depletion image of migration in SW1116 cells.

FIG. S5 ICT1 depletion does not affect cell growth and migration in normal cells. (a, b) Knockdown efficiency of ICT1 was analyzed by quantitative real-time PCR and western blot. (c) MTT assay showed that cell numbers were no difference between ICT1 depletion groups (ICT1-S1 and ICT1-S2 groups) and control group (NC group). (d) The proportion of apoptosis cell was revealed. (e) Statistical analysis of the number of migrated cells in different groups. **, p < 0.01, compared with NC group.