Fig 1: DEFA5 overexpression induces cell cycle arrest by increasing the number of S-phase cells. A, Representative samples of propidium iodide staining and flow cytometry analysis showing the cells those in S-phase, G0/G1-phase and G2/M-phase; B, The collective data showing the percentage of G0/G1-phase cells, S-phase cells and G2/M-phase cells. C, D, Western blot results showing the phosphorylation levels of cell proliferation-related marker proteins AKT and ERK. *P < .05, **P < .01 vs HA-Vec group. Data are presented as mean ± SD. The results were reproducible in 3 independent experiments
Fig 2: BMI1 knockout mimics the inhibitory effect of DEFA5 by increasing p16 and p19 expression. A, B, The protein expression levels of p16 and p19 after BMI1 knockout and/or DEFA5 overexpression. C, The capacity of cell proliferation after BMI1 knockout. **P < .01 vs HA-Vec group. Data were presented as mean ± SD. The results were reproducible in 3 independent experiments
Fig 3: DEFA5 interacts with BMI1 and suppresses its recruitment to the promoter region of CDKN2a. A, Co-IP experiment showing the direct interaction between DEFA5 and BMI1. B, The specific interaction between DEFA5 and BMI1 was confirmed by in vitro GST pull-down assay. C, Co-IP experiment indicating the interaction between HA-DEFA5 and full-length BMI1 or different truncated molecules. D, E, ChIP assay combined with quantitative PCR showing the recruitment of BMI1 to the promoter region of CDKN2a was significantly reduced by DEFA5 overexpression. D, Representative images. E, The promoter region of CDKN2a that pulled down by BMI1 antibody. F, Dual-luciferase assay demonstrating the promoter activity of CDKN2a. (G, H, I) the protein and mRNA expression levels of BIM-1, p16 and p19. **P < .01 vs HA-Vec group. Data were presented as mean ± SD. The results were reproducible in 3 independent experiments
Fig 4: DEFA5 overexpression inhibits tumor growth in vivo. A, Typical image of the subcutaneous tumors in nude mice 6 wk after inoculation. B, The size of subcutaneous tumors was measured to determine the cancer cell growth rate. *P < .05, **P < .01 vs HA-Vector group. Data are presented as the mean ± SD of 5 individual mice (n = 5)
Fig 5: DEFA5 is downregulated in gastric cancer. A, B, RNA sequencing data showing the expression level of DEFA5 in gastric cancer from TCGA database. Data from 32 normal and 375 cancer samples in gastric cancer. C, Quantitative real-time PCR experiment showing the mRNA expression of DEFA5 in the tumor tissue from gastric cancer patients. n = 12. **P < .01 vs normal control group. D, E, expression of DEFA5 in single-cell dataset (GSE134520) were analyzed using webtool: https://www.tcpaportal.org/tcpa/index.html.
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