Fig 2: Overexpressed TXNIP partially reversed the effect of Gen on the expression of senescence genes in H2O2-treated HUVECs. HUVECs were transfected with overexpressed TXNIP plasmid, treated with H2O2 or Gen alone or combination. (A, B) After transfection of overexpressed TXNIP plasmid, the relative protein expression of p16 and p21 in H2O2-treated HUVECs under 80 µg/mL Gen treatment were measured by western blot. GAPDH was used as the internal control. (C) After transfection of overexpressed TXNIP plasmid, the relative mRNA expression of p16 and p21 in H2O2-treated HUVECs under 80 µg/mL Gen treatment were determined by qRT-PCR. GAPDH was used as the internal control. All experiments were performed in triplicate and the experimental data were expressed as mean ± standard deviation (SD) (*p< 0.05, **p< 0.01, vs. NC; ?p< 0.05, ??p< 0.01, vs. NC + H2O2; #p< 0.05, ##p< 0.01, vs. TXNIP; ‡p< 0.05, ‡‡p< 0.01, vs. NC + H2O2+Gen80; ?p< 0.05, ??p< 0.01, vs. NC + Gen80). HUVECs: human umbilical vein endothelial cells; TXNIP: thioredoxin-interacting protein; NC: negative control.

Fig 3: KLF2 inhibits cell proliferation and induces cell apoptosis and cell cycle arrest(A) Alteration of KLF2 influenced the proliferation of MKN-45, BGC-823 and MGC-803 cells by CCK8 assay at different time points. KLF2 overexpression significantly inhibited MKN-45 and BGC-823 cell proliferation. Knockdown of KLF2 enhanced cell proliferation in MGC-803 cells. *** P < 0.001. (B) Percentage of apoptotic cells, including early and late stage, in KLF2 overexpressing and deficient cell lines was determined after analyzing Annexin V and PI double labeled cells through FACS. *** P < 0.001. (C) Levels of apoptosis-associated marker protein cleaved caspase 3 was determined via Western blot in KLF2 overexpressing MKN-45 cells and KLF2 deficient MGC-803 cells. (D) Comparison of cell cycle distribution in KLF2 overexpressing and deficient cell lines, based on FACS analysis. (E) Protein expression of cell cycle regulators such as p16/ CDKN2A, p27/CDKN1B and CCND1 in KLF2 overexpressing MKN-45 cells and KLF2 deficient MGC-803 cells.

Fig 4: ACSL1 induces senescence through SIRT1/p53/p21 pathways in K562 cells. (a) Western blot analysis of the expression levels of senescence related proteins (p53, p21, p16, Rb) after transfection with ACSL1 in K562 cells. (b) qRT-PCR was applied to quantify the relative mRNA expression of p21 and p16. (c) Western blot detected the expression levels of ACSL1, p53, and p21 in four groups K562 cells. (d) qRT-PCR was applied to quantify the relative mRNA expression of p53. (e) Western blot assays for ACSL1 and p53 expression in K562 NC and ACSL1 cells treated with MG132. (f) Western blot assays for p53 expression in K562 NC and ACSL1 cells treated with CHX. (g) The protein–protein networks view from STRING database showing the networks of ACSL1, p53, and SIRT1. (h) The mRNA levels of SIRT1 and MDM2 after transfection with ACSL1 in K562 cells. (i) The protein levels of SIRT1 after transfection with ACSL1 in K562 cells. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 5: In vivo experiments verifying that miR-205–3p promotes senescence of gastric cancer cells and secretes SASP factor to recruit more T cells in blood and tumors.A: Representative tumor photos of AGS xenografts following subcutaneous injection of AGS cells expressing miR-205 or negative control in nude mice. B: Tumor volume was measured on the days indicated. (n = 10, each group). C: Representative immunohistochemical staining and quantitative analysis of P16, P21 and PD-L1 expression of AGS xenograft sections of miR-205 and negative control groups. D: Representative SA-ß-gal staining and quantitative analysis of AGS xenograft sections of miR-205 and negative control groups. E: qRT-PCR assay is used to detect the mRNA levels of IL-1a, IL-1ß, IL-6, IL-8 and MMP3 in AGS xenografts of miR-205 and negative control groups. F: Representative example of CD8+CD44+ and CD8+CD69+T cells gated on live CD3+ T cells in blood and freshly dissociated xenograft tumors of miR-205 and negative control groups. **P < 0.01.