2.1. Materials and reagents
The fruiting bodies of I. obliquus were collected from Changbai Mountain, Jilin, China. Human colon carcinoma cells HT-29 were obtained from Cancer Hospital Chinese Academy of Medical Sciences, Beijing, China. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. All reagents used for cell culture were from Hyclone (USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). Tissue DNA Kit was purchased from Solarbio (Beijing, China). Annexin V-FITC Apoptosis Detection Kit was purchased from Njjcbio (Nanjing, China). Antibodies to Bcl-2, Bax, Caspase-3 and β-actin were obtained from Wanleibio (Shenyang, China).
2.2. Extraction, purification, and fraction of polysaccharides
The crude water-soluble polysaccharides were extracted from I. obliquus samples (CIOP) with distilled water at 60 °C according to the method outlined by Xu et al23. After removal of the protein by using the Sevag reagent, the CIOP solution was mixed with 95% (v/v) ethanol to reach a final concentration of 60% (v/v) and stored at 4°C overnight. After centrifugation, dialysis and freeze drying, the precipitate was termed as IOP60. 10 mg/mL IOP60 solution was prepared, filtered through a 0.22 μm membrane, and assembled with ultrafiltration centrifuge tubes (Merck Millipore Amicon™). After centrifugation and freeze drying, fractions were collected and termed as IOP60a (3 kDa ≤ molecular weight ≤ 10 kDa), IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa), IOP60c (30 kDa ≤ molecular weight ≤ 50 kDa), IOP60d (50 kDa ≤ molecular weight ≤ 100 kDa), and IOP60e (molecular weight ≥ 100 kD), respectively. Their yields were calculated based on the weight of each fraction to the weight of the total IOP60.
2.3. Cell viability analysis
HT-29 cells were seeded for 12 h in 96-well plates (2×106 cells/mL), and then treated with 1.25 mg/mL polysaccharide fraction (IOP60a, IOP60b, IOP60c, IOP60d, IOP60e) for 24 h, respectively. CCK-8 assay was used to detect the cell viability according to the manufacturer’s protocol24. All determinations were done in six duplicates. The inhibition ratio of tumor cell proliferation was calculated according to the formula below:
See formula 1 in the supplementary files.
Where As, Ac and Ab were the absorbance of treated cells, untreated cells, and the control group, respectively.
2.4. Observation of cell morphology
HT-29 cells (2×106 cells per well) were seeded in 6-well plates for 24 h and treated with different concentrations of IOP60b (0, 0.625, 1.25, 2.5, 5, 10 mg/mL) for 48 h. After that, cells were fixed with 4% formaldehyde in PBS for 10 min, and then subjected to optical microscopy (Olympus BX51, Japan).
2.5. DNA fragmentation analysis
IOP60b-treated (48 h) HT-29 cells were washed and resuspended in 1 mL PBS. Total DNA of cells was extracted according to the manufacturer’s instructions. The extracted DNA samples were run on the 1.2% agarose gel in 1×TAE buffer. After that, the gel was visualized with a UV light by Gel Imaging System and photographed.
2.6. Cell apoptosis and Cell cycle analysis by flow cytometry
The percentage of specific apoptotic cells was determined by flow cytometry according to the manufacturer’s procedure. Brieﬂy, HT-29 cells were plated in a 6-well plate (1×105 cells/mL) and treated with different concentrations of IOP60b (0.625, 1.25, 2.5, 5, 10 mg/mL) for 48 h. Cells were collected and washed twice in cold PBS, and then resuspended in 500 µL binding buffer containing 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) for 10 min in the dark, finally analyzed using Millipore guava easy CyteTM ﬂow cytometer (Millipore, USA) and Guavasoft 3.1.1 software.
Similarly, HT-29 cells were first plated in a 6-well plate (1×105 cells/mL) and treated with different concentrations of IOP60b (0.625, 1.25, 2.5, 5, 10 mg/mL) for 48 h and then used for cell cycle analysis. Cells were centrifuged at 12000 ×g for 5 min and then fixed with 70% (v/v) ethanol and stored at 4 °C overnight. The precipitate of cells was washed with PBS, treated with 100 µL RNase, and incubated for 30 min at 37 °C. After centrifugation, cells were incubated with PI at 4 °C for another 30 min in the dark. Cell cycle distributions were detected by flow cytometry and analyzed using ModFit LT 5.0 software.
2.7. Real-time quantitative RT-PCR analysis
HT-29 cells (5×105 cells/mL) were seeded in 6-well plates and treated with 5 mg/mL IOP60b for 0 h, 24 h, 48 h, 72 h, respectively. Total RNA was extracted using total RNA extraction kit (GeneMarkTM) and then reversed to cDNA by using the All-in-OneTM First-Strand cDNA Synthesis Kit (GeneCopoeia, USA) according to the manufacturer's protocol. Real-time PCR was performed with 2×Es Taq MasterMix (Dye) (CWBIO, Beijing, China). The following primers (Sangon Biotech, Shanghai, China) were used for amplification: Bcl-2 forward 5′-CAG CTG CAC CTG ACG CCC TT-3′ and reverse 5′-GCC TCC GTT ATC CTG GAT CC-3′; Bax forward 5′-TTC TGA CGG CAA CTT CAA CTG-3′ and reverse 5′-TGA GGA GGC TTG AGG AGT CT-3′; Caspase-3 forward 5′-ACA CAG TAT GGC GGC AGA G-3′ and reverse 5′-AGA CAG GCA ACA GAG CAC AT-3′; β-actin forward 5′-CCT CTA TGC CAA CAC AGT GC-3′ and reverse 5′-ATA CTC CTG CTT GCT GAT CC-3′.
2.8. Western blotting
HT-29 cells were harvested and lysed after treated with IOP60b for desired time (0 h, 24 h, 48 h, 72 h). Total proteins were extracted using a Total Protein Extraction Kit (Wanleibio, China), and the protein concentration was determined using a BCA Protein Assay Kit (Wanleibio, China) according to the manufacturer's protocol. Equal amounts of proteins were boiled for 5 min and separated by SDS-PAGE, then transferred onto a PVDF membrane. Blocking of non-specific binding was achieved by placing the membrane in a dilute solution of 5% skim milk powder in tris-buffered saline (TBS) for 1 h. After blocking, a diluted solution of primary antibody (Bcl-2, Bax, Caspase-3 and β-actin) at a concentration of 1:500 was incubated with the membrane overnight at 4 °C under gentle agitation. After that, the primary antibody was diluted with TBST wash buffers. The membrane was rinsed to remove unbound primary antibody, followed by incubation with a dilute solution of secondary antibody (HRP-labeled goat anti-rabbit Ig G) at a concentration of 1:5000 for 45 min at 37 °C. After a final wash of membrane with TBST buffers, the Electrochemilu-minescence (ECL) reagent was used to measure the chemiluminescence intensity. Tand intensities were quantitated using Gel-Pro-Analyzer software.
2.9. Statistical analysis
All the measurements and analyses were carried out in triplicate. The experimental results were presented as means of three determinations ± SD (standard deviation). Origin (version 8.5) and SPSS (version 16.0) with Tukey's multiple comparisons were used for the statistical and graphical evaluation. The statistical significance of mean differences was based on p-Values of <0.05.