METTL3-associated YTHDF1-COX-1 axis is linked to reduced antiplatelet response to aspirin in older adults

Figures

• 

  Figure 1:. Whole blood levels of m6A RNA and expression of m6A-related genes in patients with low (n = 14) and high (n = 20) aspirin response. (A) Whole blood m6A RNA was detected by colorimetric assay; (B) serum PGH2 levels were measured by ELISA; (C) serum TXB2 levels were measured by ELISA; (D) the expression of 7 genes involved in m6A modification was evaluated by real-time quantitative polymerase chain reaction (qPCR). Data are expressed as mean ± SEM (*: p < 0.05; **: p < 0.01).

• 

  Figure 2:. Effects of altered METTL3 expression on key genes involved in AA metabolism. (A–H) METTL3, YTHDF1, YTHDF2, COX-1 (PTGS1), COX-2 (PTGS2), TBXAS1, PTGIS and PTGIR mRNA expression measured by real-time qPCR; (I–O) METTL3, YTHDF1, YTHDF2, COX-1, COX-2 and TBXAS1 protein expression measured by western blotting. Data are expressed as mean ± SEM (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: p > 0.05). All experiments were repeated three times. Comparisons between the indicated groups were performed using independent-samples t-tests.

• 

  Figure 3:. Extracellular PGH2 and TXB2 levels in cell-culture supernatants over time. (A–C) PGH2 levels in cell-culture supernatants measured by ELISA at 48, 72, and 120 h; (D–F) TXB2 levels in cell-culture supernatants measured by ELISA at 48, 72, and 120 h. Data are presented as mean ± SEM. Comparisons between the indicated groups were performed using independent-samples t-tests. *: p < 0.05; **: p < 0.01; ns: not significant.

• 

  Figure 4:. METTL3 promoted the translation of COX-1 mRNA through YTHDF1-dependent mechanism. (A) Western blots of COX-1 protein after up- and downregulation of METTL3 in the presence or absence of CHX; (B) western blots of COX-1 protein following METTL3 upregulation with YTHDF1 downregulation; (C) RNA IP experiments with anti-YTHDF1 antibodies. Data are expressed as mean ± SEM (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: p > 0.05). All experiments were repeated three times. Comparisons between the indicated groups were performed using independent-samples t-tests.

• 

  Figure 5:. Effects of METTL3 on MEG-01 cell proliferation and apoptosis. (A) Proliferation of MEG-01 cells following METTL3-overexpressing or METTL3-knockdown lentiviral transfection for 48 h, evaluated using the CCK-8 assay; (B) proliferation of MEG-01 cells following METTL3-overexpressing or METTL3-knockdown lentiviral transfection for 72 h evaluated using the CCK-8 assay; (C) apoptotic status 24 and 48 h after transfection with METTL3-overexpressing or METTL3-knockdown lentivirus determined by Annexin V-EGFP/PI staining and flow cytometry. Data are expressed as mean ± SEM (*: p < 0.05; ns: p > 0.05). All experiments were repeated three times. Comparisons between the indicated groups in panels (A) and (B) were performed using independent-samples t-tests.

• 

  Figure 6:. Schematic diagram illustrating the mechanism of the METTL3-mediated YTHDF1-COX-1 axis in reducing the antiplatelet efficiency of aspirin in older adults.