Cell lines and reagents
The HCT-116 and HT-29 human CRC cell lines were purchased from ATCC (Manassas, VA, USA). RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin was used to culture both kinds of cells. Cells were cultured in a 37°C incubator with 5% CO2. Sodium butyrate (NaB) was purchased from Aladdin (Shanghai, China). Recombinant human IL-6 protein was obtained from R&D Systems (Minneapolis, MN, USA).
Cell viability assay
An enhanced Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China) was used to measured cell viabilities. HCT-116 cell and HT-29 cell were cultured in 96-well plates with 0-10 mM NaB at a density of 5000 cells per well for 0-36 hours. Then, CCK-8 assay solution was added to each sample. All cells were incubated for 1.5 hours in the dark. The absorbance was assessed at 450 nm. For each condition, 6 independent biological duplicates were assessed.
EdU cell proliferation assay
The cell proliferative ability was assessed by an EdU cell proliferation kit (Beyotime). According to the instructions, EdU working solution was added to cells pretreated with 5 mM NaB for 24 hours. Then, all samples were cultured in an incubator for 2 hours and fixed for 15 minutes. All cells were stained with EdU and Hoechst 33342. Cells were counted and imaged with an Olympus FSX100 microscope (Olympus, Tokyo, Japan).
Apoptosis assay
Apoptosis was assessed by fluorescence-activated cell sorting (FACS) analysis according to the instructions of the Annexin V-FITC detection kit (KeyGEN BioTECH, Nanjing, China). HCT-116 and HT-29 cells were treated with 5 mM NaB or 10 mM NaB for 24 hours. Then, all cells were collected and incubated with FITC-conjugated Annexin V and propidium iodide (PI) dye. Apoptosis was measured by flow cytometry (BD Biosciences Franklin Lakes, NJ, USA). Results were analyzed with CellQuest software.
Colony formation assay
Cells in logarithmic phase were collected and resuspended in RPMI 1640 medium containing 5 mM NaB. Then, cells were transferred to 6-well plates (400 cells per well) and incubated for 14 days. All media were replaced daily. Then, colonies were fixed for 15 minutes and stained with 1% Giemsa stain solution (Solarbio, Beijing, China) for 30 minutes. All colonies were counted.
Wound healing assay
HCT-116 cell and HT-29 cell were cultured in 12-well plates. At 100% confluence, scratches of equal widths were made in the cell monolayers with sterile 10-µl pipette tips. After washing with PBS, all cells were cultured in FBS-free RPMI 1640 medium containing 5 mM NaB for 24 hours. Images were acquired with an Olympus FSX100 microscope (Olympus). The wound closure percentage was calculated with ImageJ software (USA).
Transwell assay
Transwell chambers (Corning Costar, Cambridge, MA, USA) with 8-µm pores were used for this assay. For the cell migration assay, cells were cultured in FBS-free RPMI 1640 medium overnight and transferred to the upper chambers (105 cells per well) the next day. Then medium with 10% FBS and 5 mM NaB was added to lower chambers. 24 hours later, the filter membranes were fixed for 15 minutes after washing with PBS and stained with 1% Giemsa stain solution for 30 minutes. Then, the cells remaining on the upper surface of membranes (non-migrated cells) were gently removed with cotton swabs. The cells remaining on the lower surface (migrated cells) were counted and imaged. For the cell invasion assay, filter membranes precoated with a 1:6 dilution of Matrigel (BD Bioscience, San Diego, CA, USA) were used instead of uncoated filter membranes. The other steps were performed the same as in the cell migration assay.
Western blot analysis
Cells were collected for protein extraction after treated with 5 mM NaB for 24 hours and then 25 ng/ml IL-6 for 30 minutes. RIPA lysis buffer containing protease and phosphatase inhibitors was used to extract cellular proteins. Proteins were separated by SDS-PAGE and were then transferred to PVDF membranes and probed with specific primary antibodies and secondary antibodies. An enhanced chemiluminescence detection kit (Beyotime) was used to visualize the bands. Antibodies specific for gp80 (sc-373780, 1:100) and gp130 (sc-376280, 1:100) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies specific for STAT3 (#9139, 1:1000), JAK2 (#3230, 1:1000), p-STAT3 (#9145, 1:1000), p-JAK2 (#3771, 1:1000) and TRAF5 (#41658, 1:1000) were obtained from CST (Beverly, MA, USA). The anti-GAPDH antibody (BM1623, 1:1000), anti-β-actin antibody (BM0627, 1:1000), anti-α-tubulin antibody (BM1452, 1:1000), anti-rabbit IgG-HRP antibody (BA1054, 1:5000) and anti-mouse IgG-HRP antibody (BA1050, 1:5000) were purchased from Boster Biological Technology (Wuhan, China).
Protein stability assay
Cells were pretreated with 5 mM NaB for 24 hours. Then, 0.5 hours, 1 hour and 2 hours before cells were collected, 30 µg/ml cycloheximide (CHX; MCE, Monmouth Junction, NJ, USA) was added to all cells to block protein translation. The gp130 protein level was assessed by western blot assay.
TCGA database and analysis
TRAF5 gens were analyzed by GEPIA web tools (http://gepia.cancer-pku.cn/) based on TCGA database.
SCFAs Assay
SCFAs were assessed from fecal samples homogenized in buffers. Suspension was centrifuged and the supernatant was collected for test. A gas chromatography and a fused silica capillary column (NukonTM, Bellefonte, PA, USA) were used for this assay.
Cell transfection
HT-29 cells were transfected with siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the instruction. Briefly, cells were seeded in 6-well plates at the density of 2 × 104 cells per well the day before transfection. Then cells were transfected with 20 nM siRNA the next day. All mediums were replaced by culture medium 6 hours after transfection and the transfection efficiency was assessed by fluorescence microscope after 48 hours. Then transfected HT-29 cells were used for experiments according to protocols. The TRAF5 siRNA kit was purchased from Ribobio (Guangzhou, China).
Immunoprecipitation
For this assay, all cells were lysed in NP-40 buffers and centrifuged at 12000 rpm for 10 minutes. Proteins were immunoprecipitated from supernatants with primary antibodies immobilized on protein G agarose beads overnight at 4 °C. Then all proteins were collected for western blot.
RNA extraction and qRT-PCR
Cells treated with 5 mM NaB for 24 hours and 25 ng/ml IL-6 for 30 minutes were collected for RNA extraction. RNA was extracted with a RNeasy Mini Kit (Qiagen, Valencia, USA) and then reverse transcribed to cDNA. The mRNA expression levels were assessed by a VII A7 real-time PCR system (Applied Biosystems, Foster, CA, USA). The primers were listed in Supplementary file 3: Table S1.
Immunofluorescence
All cells were treated with 5 mM NaB for 24 hours and 25 ng/ml IL-6 for 30 minutes and fixed for 15 minutes and blocked with 5% goat serum for 1 hour. Then, cells were incubated with the anti-p-STAT3 antibody (CST, 1:200) overnight at 4°C and with a Cy3-conjugated secondary antibody (Beyotime, 1:500) for 1 hour the next day. Images were acquired with a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
Mouse xenograft tumor model
All animal experiments were manipulated according to guidelines approved by the Animal Care Committee of Zhejiang University School of Medicine. A total of 106 HT-29 cells resuspended in 200 µl FBS-free RPMI 1640 medium were injected into BALB/c athymic nude mice (6-week-old, female) subcutaneously. Tumor volumes were measured every two days. When the tumor volume reached 60-80 mm3, mice were divided into different groups randomly. For dietary fibers experiments, the low-fiber diet (LFD) group was fed with low fibers food (20% fiber) and the high-fiber diet (HFD) group was fed with high fibers food (70% fiber) for 2 weeks. All diets were purchased from Test Diet (Richmond, IN). After 2 weeks, all animals were sacrificed, and tumor tissues and normal peritumoral tissues were collected. All tissues were frozen in liquid nitrogen and fixed with 4% paraformaldehyde for western blot analysis, hematoxylin and eosin (HE) staining and immunofluorescence staining. For butyrate experiments, the control group was intraperitoneally injected with normal saline every day while the NaB group was intraperitoneally injected with 200 mg/kg NaB. Other steps were performed the same as above.
Statistical analysis
Data were analyzed by SPSS software (Chicago, IL, USA) and GraphPad Prism 7.0 (San Diego, CA, USA). The differences between groups were calculated by ANOVA and Student’s t-test. All data are presented as the mean ± SEM. P < 0.05 was considered significant.