br Annexin V FITC PI
2.7. Annexin V-FITC/PI staining
Apoptosis in the CRC cells was determined by Annexin V/PI staining as previously described . Briefly, 2 × 105 cells were seeded per well in 6-well plates and cultured with various concentrations of AIF for 48 h. The cells were then stained with Annexin V-FITC/PI kit (#C1062, Beyotime Biotechnology Shanghai, China) according to the manufac-turer's instructions. The percentage of apoptotic cells were analyzed by flow cytometry (Beckman Coulter Inc., Miami, FL, USA).
2.8. Immuno-fluorescence staining
HCT-116 and SW480 cells were seeded into 24-well plates, with each well containing a glass coverslip, at the density of 2 × 104 cells/ well. Following incubation with different concentrations of AIF for the indicated durations, the coverslips were retrieved and air-dried, and the adherent cells were fixed with 2% paraformaldehyde for 30 min. The cells were rinsed in TBS, permeabilized with methanol at −20 °C for 1 min, and rinsed again before blocking with 1% bovine serum albumin (BSA) and 0.2% Tween-20 (TTN) in TBS for 20 min. The cells were then incubated for 2 h with anti-p-γH2AX (Ser-139) mAb (1:500 in TTN; Upstate, Lake Placid, NY). The coverslips were washed and incubated with FITC conjugated anti-mouse goat F (ab′) 2 fragment (1:200 in TTN; DAKO, Carpinteria, CA) for 1 h at room temperature. After rinsing the coverslips, they were immersed in 0.05 mg/ml DAPI for 15 min, rinsed again, and mounted using 10 μl Fluorogard (Bio-Rad) and sealed. Eight hundred randomly selected cells were counted per sample, and cells with three or more γH2AX foci of any size were classified as positive.
2.9. Western blotting
The expression level of specific proteins was evaluated by Western blotting as previously described . Briefly, following 48 h treatment with AIF, the cells were lysed and the protein concentration in the cell lysates was measured. Equal amounts of proteins per sample were re-solved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) mem-branes. The blots were subsequently incubated overnight with the pri-mary Cyclosporin H against RAD51, γH2AX, pro-caspase-3, cleaved cas-pase-3, Bax, Bcl-2 and GAPDH (internal control) at 4 °C. After incubation with peroxidase-conjugated secondary antibodies, the im-muno-reactive bands were visualized by enhanced BeyoECL Star (no. P0018AM, Beyotime Biotechnology Shanghai, China) according to the manufacturer's instructions.
2.10. Proteomics analysis
iTRAQ-based proteomics analysis was quantitatively analyzed by Shanghai OE Biotech (Shanghai, China) as described previously . In briefly, Following AIF treatment, cells were dissolved in lysis buffer and labelled with iTRAQ labelling reagents. The samples were subjected to LC analysis and tandem mass spectrometry analysis. Protein
identification and relative iTRAQ quantification service were provided by Oebiotech (Shanghai, China). The cutoff value for the differentially expressed proteins was adjusted to p < 0.05 and the fold change was set to > 1.5 or < 0.5.
2.11. In vivo CRC xenograft model establishment
The Institutional Animal Care and Use Committee at Puyang Oilfield General Hospital approved all animal experiments in this study. Eight week-old male athymic BALB/c nu/nu mice were housed in pathogen-free conditions and given sterile food and water ad libitum. After ac-climatization, the mice were each injected with 107 HCT-116 cells in their left flanks. Twenty-one days after implantation, the mice were randomized into 3 groups (6 mice/group) and injected i.p. as follows:
(ii) AIF (25 mg/kg/day dissolved in vehicle), and (iii) AIF (50 mg/kg/ day). The body weight pf the mice and the tumor volume were mea-sured twice every week until the 24th day. The mice were sacrificed and the tumors were resected and weighed. The tumor tissues were processed for histopathological evaluation by hematoxylin and eosin (H &E) staining. In situ apoptosis in the tumor tissues was analyzed by TUNEL staining (Biyuntian, Wuxi, China) according to the manufac-turer's instructions.
2.12. Statistical analysis
Data are presented as means ± SD from three independent ex-periments. All statistical analyses were performed using GraphPad Prism 7.0 and SPSS 17.0 software. One-way ANOVA followed by Dunnett's t-test was used to compare multiple groups. The relationship between RAD51 levels and the overall survival of CRC patients was analyzed by Kaplan-Meier and log-rank test. A p value < 0.05 was considered statistically significant.
3.1. AIF inhibits CRC cell proliferation and promotes apoptosis
The anti-cancer effect of AIF was tested on two well established CRC cell lines, HCT-116 and SW480. The viability of both lines was sig-nificantly reduced following exposure to AIF in a time and dose de-pendent manner, compared to the vehicle control group (Fig. 1A). Consistent with above results, fewer colonies were formed by the AIF-treated cells compared to the control cells (Fig. 1B). Since chromatin condensation is a hallmark of apoptosis , we stained the cells with the DNA-specific Hoechst 33258. As shown in Fig. 1C, a significantly higher number of AIF-treated cells had chromatin condensation com-pared to the vehicle group. In addition, Annexin V/PI staining showed that AIF increased the percentage of apoptotic cells in a dose dependent manner – from 5.2% (HCT-116) and 4.7% (SW480) in the vehicle group to 13.2% and 19.2% in the 5 μM AIF-treated cells, and 22% and 30% in the 10 μM AIF-treated cells (Fig. 1D). Interestingly, the SW480 cells