2.10 Immunofluorescence
Cells cultured on glass coverslips were washed with PBS for 3 times, fixed with 4% formaldehyde-PBS solution for 10 min, and permeabilized with 0.2% Triton X-100 PBS solution for 10 min. After that, the cells were washed with PBS and incubated with a primary antibody against ING5 (Abcam, UK), APC (Novus, USA) and β-catenin (CST, Massachusetts, USA) at 4℃ overnight. They were then washed with PBS and incubated with Alexa Fluor 350 rabbit anti-goat IgG (Bioss, China), CY3 goat anti-mouse IgG(H + L) (Beyotime, China). The results were observed under a confocal laser scanning microscope (BX53, Olympus, Japan).
2.11 Luciferase reporter assay
To evaluate the transcriptional activity of β-catenin/TCF-4 and NF-κB in the cells, we cotransfected T-cell factor-4 (TCF-4)-dependent luciferase reporter gene plasmids (pGL3-OT), negative control plasmids (pGL3-OF) (Beyotime, China), and Renilla luciferase reporter gene plasmids (pRL-TK) as an internal control by applying Lipofectamine 3000. The firefly and Renilla luciferase activity of cells was measured 48 h after transfection with a dual-luciferase reporter assay kit (Beyotime, China) as recommended by the protocol. TCF-mediated gene transcription activity was evaluated by the related luciferase unit (RLU) on a microplate reader (Infinite® 200 Pro, Tecan, Mannedorf, Switzerland). NF-κB promoter activity was determined as described above with pGL6-NF-κB-luc plasmids (Beyotime, China) and control plasmids.
2.12 Mouse studies
Six-week-old female nude mice (BALB/c-nu) (Beijing HFK Bioscience, China), villin-cre mice (Jackson Lab, USA), and CRISPR/Cas9-ING5Loxp/Loxp mice based on the C57BL/6J strain (made by Shanghai Biomodel Organism Science & Technology Development Co., Ltd., China) were bred and maintained in specific pathogen-free conditions.
We crossed villin-cre mice with CRISPR/Cas9-ING5Loxp/Loxp mice to obtain villin-cre/ ING5Loxp/Loxp mice. To chemically induce colorectal tumors, we injected azoxymethane (AOM, 10 mg/kg) intraperitoneally and administered 2.5% dextran sodium sulfate (DSS) in sterile distilled water orally to villin-cre/ING5Loxp/Loxp mice (n = 24, 16–18 g, 8 weeks old) and wild-type (WT) mice (n = 23, 16–18 g, 8 weeks old) mice for 6 weeks. To evaluate the proliferation ability and chemotherapy resistance of cells in vivo. A total of 1× 106 cancer cells per mouse (6–8 weeks old, 6 mice per group) were injected subcutaneously to establish xenograft models. Tumor volume was measured using calipers and calculated as follows: length × width × depth × 0.52. Treatment with cisplatin was performed by intratumoral multiple-point injection (10 mg/kg) when the tumor grew to a diameter length of 5 mm. Mice were euthanized by CO2 asphyxiation, and tumors were removed and fixed in 4% formaldehyde.
2.13 PCR
DNA was extracted from tail tips by proteinase K digestion and phenol/chloroform extraction. To confirm the conditional knockout of the ING5 genetic phenotype, we performed standard PCR using DNA from tail tips targeting ING5 and cre with Taq Supermix polymerase (Magic-Bio, China). The primers for villin-cre/ING5Loxp/Loxp mouse identification were P1: 5’-TCCTCTCTGGTTCAG CAGA-3’, P2: 5’-CTAAATGAGTACACTTACAC-3’, P3: 5’-ATCACTACCTAGATGAGATA-3’, P4: 5’-AGAGCAGTCAGTGCTCCCAA-3’ and P5: 5’-AATGAGCAGAAGAG GACG AG-3’.
Total RNA was extracted from colorectal cancer cells. AMV reverse transcriptase and random primers (Takara, Japan) were employed to synthesize cDNA from the total RNA. Real-time PCR was performed using the SYBR Premix Ex Taq™ II kit (Takara, Japan). Oligonucleotide primers were designed according to GenBank and are shown in Supplementary Table 1.
2.14 Co-immunoprecipitation (Co-IP)
Protein extracted from cells was precleared with protein A-Sepharose beads (Roche, USA) for 60 min. The samples were centrifuged at 14000 ×g for 10 min to remove nonspecifically bound proteins. A 1-mL protein sample (1 µg/mL) was incubated with 5 µg primary antibody on a rotator overnight. After that, 100 µL Protein A Sepharose beads (50% slurry) were added to the protein sample and centrifuged at 4°C overnight. The beads were washed three times with lysis buffer containing 1% NP40. The precipitate was heated with 50 µL 2×SDS loading buffer at 100°C for 5 min. The samples were centrifuged to remove the beads, and the supernatant was prepared for Western blot analysis.
2.15 Western blotting
The denatured protein sample was separated by SDS-PAGE and transferred to a PVDF membrane (Promega, USA), which was then blocked in 5% milk for 1 h. The membrane was incubated with primary antibody (Supplement Table 2) for immunoblotting. Then, the cells were rinsed with TBST for 3 times and incubated with secondary antibody for 1 h. The bands were visualized by ECL detection reagents (Thermo Fisher, USA). Quantifications were conducted in Image J software (NIH, USA).
2.16 Immunohistochemistry (IHC)
Formalin-fixed, paraffin‐embedded tissue sections were deparaffinized and rehydrated 3 times, and antigen retrieval was performed with antigen retrieval solution (DAKO, Japan) in a microwave oven for 20 min. Endogenous peroxidase activity and antigen sites with nonspecific binding were blocked by 3% hydrogen peroxide (H2O2) and then 5% bovine serum albumin (BSA) for 30 min, respectively. Then, the sections were incubated with primary antibody (Supplementary Table 2) at room temperature for 1 h. After rinsing with PBS 3 times, the sections were incubated with specimen-specific IgG-conjugated HRP (DAKO, USA) at room temperature for 2 h. Diaminobenzidine (DAB) was used to visualize the immunocomplexes. After staining with Mayer’s hematoxylin, the sections were dehydrated, cleared and mounted. Images were acquired by a microscope (Nikon, Japan).
2.17 Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
The TUNEL assay was used to detect apoptotic cells undergoing extensive DNA degradation during apoptosis. For this purpose, the In Situ Cell Death Detection Kit (Roche, USA) was employed according to the recommendation. Omission of the working strength of the TdT enzyme was considered a negative control.
2.18 Bioinformatics analysis
RNAseq data in level 3 HTSeq-FPKM format was downloaded from the TCGA COAD/READ project. TPM format and log2 data conversion were conducted. The clinicopathological and prognostic significance of ING5 was analyzed and visualized by pROC package (version 1.17.0.1), rms package (version 6.2-0), survival package (version 3.2–10), survminer package (version 0.4.9) and ggplot2 package (version 3.3.3) of R software.
2.19 Statistical analysis
All experiments were repeated 3 times, and data are presented as the means ± SD. Unpaired two-tailed Student’s t test and Mann-Whitney U test were employed to differentiate the means. Growth curves of body weight in mice were compared two-way ANOVA, followed by Tukey or Bonferroni multiple comparisons test to determine body weight differences in each week. Statistical analyses were performed and visualized by GraphPad Prism 8 software (La Jolla, CA, USA).