Cell lines, cell culture, and transfection

Human CRC cell lines (HCT116 and LoVo) were purchased from the Shanghai Branch Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cells were cultured in DMEM (Gibco, ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% antibiotics (penicillin/streptomycin, Gibco) at 37 °C with 5% CO2. The cell lines were tested for potential mycoplasma contamination by PCR and were confirmed to be mycoplasma-negative. In subsequent experiments, the cells were treated with MG-132 (MedChemExpress, NJ, USA) or cycloheximide (CHX, MedChemExpress) at the indicated concentrations in DMEM with 10% FBS. DMSO was used as a negative control. For chemotherapy susceptibility experiments, cells were treated with cis-platinum (MedChemExpress) for 24 h or 5-Fu (MedChemExpress) for 48 h at indicated concentrations in DMEM with 10% FBS.

siRNA (sequence details in Supplementary Table S2) was transfected using Lipofectamine RNAi Max (Invitrogen, ThermoFisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, the appropriate amount of siRNAs was diluted in 100 µL Opti-MEM (Gibco, USA) and mixed with 5 µL Lipofectamine RNAi Max. The mixture was incubated at 25 °C for 20 min and then added to cell culture media. Cells were collected for further experiments after 48 h. The plasmids were constructed using pcDNA3.0. Plasmids were transfected using Lipofectamine 3000 (Invitrogen). Briefly, 150 µL Opti-MEM was used to dilute 2 µg plasmids with 5 µL p3000 and 5 µL lipo3000 separately. The plasmids and lipo3000 solution were mixed and incubated at room temperature for 15 min. The mixture was added to six-well plates, and the cells were collected after 48–72 h.

For lentivirus transfection, the PBX1 overexpression lentivirus and the negative control, purchased from iGene Biotechnology Co., Ltd. (Columbia, MD, USA), were added to cultured cells at MOI = 1:10. Polybrene (5 µL) was added to the medium. Finally, cells were cultured with 2 µg/mL puromycin to select positively transfected cells. The transfection efficiency was detected by qPCR (Primers details in Supplementary Table S3) and western blotting (Antibodies details in Supplementary Table S4).

CCK-8 assay

Cells were seeded in 96-well plates at 2000 cells/well for the CCK-8 assay. After 0, 24, 48, 72, and 96 h of planting, relative cell numbers were evaluated using 10% CCK-8 (SolarBio, Beijing, China) diluted in standard culture media for 2 h. Quantification was performed on a microtiter plate reader (ALLSHENG, Hangzhou, Zhejiang, China) at a UV wavelength (λ) of 450 nm.

Cell cycle assay

The cells were cultured in six-well plates at 60–70% fusion degree. Before the experiments, FBS-free medium was added for 12 h to pause the cell cycle. We then used complete media for 8 h and collected cells in pre-cooled 70% ethanol overnight. The cells were then centrifuged at 2000 rpm and washed twice with PBS. Finally, the cells were stained using the Cell Cycle Kit (Yishan, Shanghai, China) for 30 min and detected with CytoFLEX (Beckman Coulter, Brea, CA, USA).

EdU stain analysis

The population of DNA-replicating cells was assessed using an EdU detection kit (RiboBio, Guangzhou, China). Briefly, the cells were pre-seeded in a 96-well plate for 12 h. Then, EdU staining solution was added to the media and incubated for 2 h. After washing the additional EdU dye, Hoechst stain was used as per the manufacturer’s instructions. Images were obtained using a fluorescence microscope (BX63; Olympus, Tokyo, Japan). The EdU-positive rate was calculated as the ratio of the number of EdU-positive cells to the number of Hoechst-stained cells.

Transwell analysis

To detect cell migration, 100,000 cells in 400 µL FBS-free media were plated on the top chambers of Transwell clear polyester membrane inserts (Corning Costar, New York, USA). Culture medium containing 10% FBS was applied to the bottom. After 16–72 h, cells were stained with crystal violet and counted under a microscope (Olympus, Tokyo, Japan). For the invasion assay, Transwell clear polyester membrane inserts were pre-coated with 10% Matrigel (Corning Costar) on the top chambers for 2 h. The subsequent steps were the same as those used for the migration analysis.

Animal models

Specific Pathogen-Free male BALB/c nude mice (5–6 weeks old) were purchased from Biotechnology Co., Ltd. (Beijing, China), and maintained in specific pathogen-free facilities. This study was approved by the institutional ethical board of the First Affiliated Hospital of Sun Yat-sen University. For the tumor xenograft models, 5 × 106 cells were subcutaneously injected into the right axilla of nude mice (n = 6 per group). The tumor volume was monitored every other day (volume = length × width2 × 1/2). When the largest tumor diameter reached 1 cm, mice were euthanized via cervical dislocation. The tumors were weighed, imaged, fixed in 4% paraformaldehyde, or frozen for further analysis. For the lung metastasis model, 1 × 106 cells were injected intravenously into the tail vein of the nude mice (n = 6 per group). After 8 weeks, the mice were euthanized by cervical dislocation, and the lungs were resected, photographed, and fixed in 4% paraformaldehyde for further analysis. One animal of lung metastasis model was excluded because of unexpected death before harvested.

Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded specimens of xenograft models were cut into 3-μm-thick sections and mounted onto adhesion microscope slides. After being deparaffinized, antigen retrieval was performed with an autoclave in 0.01 mol/L EDTA buffer (pH 8.0). Sections were incubated with 3.0% hydrogen peroxide (H2O2) solution for 20 min and then incubated with 5% goat serum diluted in PBST (0.3% Triton X-100 in PBS; Gibco) at 25 °C for 30 min to block non-specific staining. Next sections were incubated with the anti-PBX1 (1:500, rabbit, 18204-1-AP, Proteintech) or anti-Ki-67 (1:500, mouse, 27309-1-AP, proteintech) at 4 °C overnight. After rinsed and sequential 1-h incubations with horseradish peroxidase-conjugated secondary antibody (G1214; Servicebio, Wuhan, Hubei, China), targeted protein was visualized using liquid DAB (Servicebio). Finally, all slides were counterstained with hematoxylin (Solarbio) as instruction.

hematoxylin–eosin staining (H&E)

The slides were prepared, deparaffinized and hematoxylin stained as described in IHC stain. Then eosin was counterstained for 2 min and washed with ddwater twice.

RNA sequencing

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Then, eukaryotic mRNA was enriched by Oligo(dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM Magnetic Kit (Epicentre, Madison, WI, USA). After fragmented into short fragments, mRNA was reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP, and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end-repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq2500.

Spindle function assay

Before the experiments, 10 µmol/L MG-132 was added to the medium for 2 h to block the division of cells in metaphase. The cells were fixed with 4% formaldehyde (Baishi, Tianjin, China), and quenched with glycine (#G8200, Solarbio) at room temperature. The cells were then incubated with diluted 5% goat serum and incubated with α-tubulin antibody (1:500, rabbit, C1050, Beyotime Biotechnology, Shanghai, China) for 2 h at room temperature. The cells were then fixed on the object slide using SlowFade Gold Antifade Mountant (Sigma-Aldrich, Merck Millipore, Darmstadt, Germany). Images were acquired using a BX63 microscope (Olympus).

Chromatin immunoprecipitation (ChIP)

ChIP was performed using a ChIP assay kit (SimpleChIP® Plus Sonication Chromatin IP Kit (#56383, Cell Signaling Technology, MA, USA). Briefly, 5 × 106 CRC cells were fixed with 1% formaldehyde and quenched with glycine (#G8200; Solarbio) at room temperature. After resuspension in lysis buffer (Cell Signaling Technology, Danvers, MA, USA), the samples were sonicated to break chromatin. Next, the chromatin solution was immunoprecipitated using anti-IgG (DIA-AN, Q6004; Wuhan, Hubei, China) and anti-PBX1 (Abnova, H00005087-M01; Tsukiji, Tokyo, Japan). Immunoprecipitated DNA was purified by column collection and analyzed by qPCR. The enrichment percentage was calculated using the relative quantification method (CT^ChIP − CT^input).

Luciferase reporter assay

The DCDC2 regulator region was amplified and subcloned into the GV-148 vector (iGene Biotechnology Co., Ltd.) for the luciferase assay. Cell samples were co-transfected with the GV-148 vector and the indicated transfection plasmids for 48 h, and then subjected to the Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Statistical analysis

Overall survival (OS) is defined as the interval between the first radical resection to the date of death from any cause or to the date of the last follow-up visit. Kaplan–Meier analysis was used to analyze the survival fraction. All statistical values were calculated using Statistical Product and Service Solutions (SPSS) 22.0 (Chicago, IL, USA). The variance similarities between the groups were analyzed by Bartlett test. The data are the means ± SEM of at least three independent experiments. Experimental studies were analyzed by independent sample t test (two-tailed) to compare two groups and by one-way ANOVA followed by Tukey’s test to compare multiple groups. Kaplan–Meier analysis and log-rank tests were used to evaluate differences in patient survival. Statistical significance was set at P < 0.05.