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. 2024 Sep 30;13(19):e034470. doi: 10.1161/JAHA.124.034470

Figure 2. METTL 3 regulates PASMC pyroptosis.

Figure 2

A, METTL3 knockdown decreased the m6A levels in the total RNA from PASMCs, as revealed by the m6A dot blot assay. B, Knockdown of METTL3 by siRNA increased the LDH activity in PASMCs, as detected by an LDH release kit (nitrocellulose‐Si‐METTL3, n=6; Hyp, n=6; Si‐METTL3‐1, n=6; Si‐METTL3‐2, n=6). C, D, METTL3 siRNA increased the mRNA and protein levels of NLRP3, caspase‐1, interleukin‐1β, interleukin‐18, and ASC, similar to those in PASMCs exposed to hypoxia, as detected by western blot and qRT‐PCR (nitrocellulose‐Si‐METTL3, n=6; Hyp, n=6; Si‐METTL3‐1, n=6; Si‐METTL3‐2, n=6). E, Knockdown of METTL3 by siRNA reduced the number of positive PI‐stained PASMCs. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F, Concentration of interleukin‐18 and interleukin‐1β in cell supernatants as detected by ELISA kit (nitrocellulose‐Si‐METTL3, n=3; Hyp, n=3; Si‐METTL3‐1, n=3; Si‐METTL3‐2, n=3). G, METTL3 overexpression increased the m6A levels in total RNA from PASMCs, as revealed by the m6A dot blot assay. H, METTL3 overexpression reversed the upregulated LDH activity induced by the exposure of PASMCs to hypoxia for 48 h, which was detected by an LDH release kit (Vector, n=7; HYP+Vector, n=7; HYP+METTL3, n=7). I, J, METTL3 overexpression reversed the increased mRNA and protein levels of NLRP3, Caspase‐1, interleukin‐1β, interleukin‐18, and ASC induced by the exposure of PASMCs to hypoxia for 48 h, as detected by qRT‐PCR and western blot, respectively (Vector, n=6–7; HYP+Vector, n=6–7; HYP+METTL3, n=6–7). K, METTL3 overexpression reversed the increased positive PI staining induced by the exposure of PASMCs to hypoxia for 48 h. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). Scale bars=100 μm. Each data point in the figure represents a unique biological replicate. L, Concentration of interleukin‐18 and interleukin‐1β in cell supernatants as detected by ELISA kit (Vector, n=3; HYP+Vector, n=3; HYP+METTL3, n=3). Statistical analysis was performed with 1‐way ANOVA. The data are presented as the mean±SD. *P<0.05, **P<0.01, ***P<0.001. ASC indicates apoptosis‐associated speck‐like protein containing a caspase recruitment domain; Hyp, hypoxia; LDH, lactate dehydrogenase; m6A, N6‐methyladenosine; METTL, methyltransferase‐like; NLRP3, nucleotide‐binding oligomerization segment‐like receptor family 3; Nor, normal; PASMC, pulmonary artery smooth muscle cell; PI, propidium iodide; qRT‐PCR, quantitative real‐time polymerase chain reaction; si‐METTL, small interfering methyltransferase‐like; and siRNA, small interfering RNA.