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. 2020 Nov 5;11(3):857–880. doi: 10.1016/j.jcmgh.2020.10.015

Figure 9.

Figure 9

CXCR2 forms a positive feedback loop with p53 to enhance the cellular senescence of gastric epithelial cells. (A) Western blot analysis of CXCR2 expression in AGS and 293T cells transfected with lentiviruses encoding p53. (B) qPCR analysis of CXCR2 mRNA expression in 293T cells transfected with p53 (CDKN1A mRNA, encoding p21, was used as a positive control, means ± SD, unpaired t test). (C and D) qPCR and Western blot analyses of CXCR2 expression in AGS cells treated with Nutlin-3 in a time-dependent and dose-dependent manner. (E) Western blot analysis of p53 and CXCR2 expression in AGS cells transfected with p53 small interfering (si)RNA followed by radiation treatment. (F) Western blot analysis of CXCR2 in the HGC27 and KATO III cell lines after X-ray exposure. (G) SA-β-gal and BrdU staining of senescent HGC27 and KATO III cells after X-ray exposure. Scale bar: 50 μm. Repeated once. (H) Diagram showing the p53 transcription response element (RE) predicted by JASPAR and PROMO. (I) Enrichment scores of different REs obtained from ChIP analysis (means ± SD, unpaired t test) and exemplary DNA agarose gel electrophoresis images from 2 independent repeats. (J) Left: diagram of wild-type (Wt) and 4 mutant (Mut) reporters. Each predicted RE was deleted in 1 mutant reporter. Right: Relative luciferase activity of different promoters in AGS cells treated with Nutlin-3 (10 μmol/L) for 48 hours (means ± SD). (K) Relative luciferase activity of different promoters in 293T cells transfected with vector or wild-type p53 plasmid. Means ± SD. Luciferase data are representative of 3 independent repeats. (L) AGS cells were first infected with PMSS1 (MOI, 100) for 3 hours and then eradicated by antibiotics (penicillin and streptomycin) for 3 hours. Samples then were harvested, analyzed by Western blot, and compared with samples with active H pylori infection for 6 hours. Right: Representative images of AGS cells with H pylori infection and after H pylori eradication are shown. ∗P < .05. ∗∗P < .01. ∗∗∗P < .001. ∗∗∗∗P < .0001. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HF, His-Flag; LUCI, luciferase; NC, negative control.