Fig 2: METTL3 drove PM2.5-induced mitophagy-dependent ferroptosis through m6A-dependent stabilization of PINK1 mRNA. (A, C) Western blotting determined the protein levels of Pink1 and Parkin in mouse lung tissues. (B, D) Western blotting determined the protein levels of PINK1 and PARKIN in Beas-2B cells. (E, F) qRT-PCR analysis of PINK1 and PARKIN mRNA levels in Beas-2B cells. (G) Scatter plots showing the correlation between mitophagy pathway and ferroptosis following PM2.5 exposure. (H) MTT tested the cell viability after inhibiting PINK1. (I, K) Representative images of lipid peroxidation after staining LiperFluo Probe in Beas-2B cells following silencing of PINK1; scale bars = 10 μm. (J, L) Representative images of ferrous ions after staining FerroOrange Probe in Beas-2B cells following silencing of PINK1; scale bars = 10 μm. All data presented in this study are representative of at least three independent experiments. Data are presented as the mean ± SEM. PM2.5, particulate matter ≤2.5 μm; m6A, N 6-methyladenosine.

Fig 3: METTL3 drove PM2.5-induced mitophagy-dependent ferroptosis through m6A-dependent stabilization of PINK1 mRNA. (A, B) qRT-PCR analysis of the mRNA levels of PINK1 and PARKIN in Beas-2B cells with METTL3 overexpression. (C) Western blotting analysis of PINK1 and PARKIN expression in Beas-2B cells with METTL3 overexpression. (D, E) Immunofluorescence analysis of PINK1 and PARKIN levels in Beas-2B cells with METTL3 silence; scale bars = 10 μm. (F, G) Quantitative analysis of the mean fluorescence intensity of PINK1 and PARKIN. (H) Immunofluorescence analysis of PINK1 and PARKIN in lungs in each group; scale bars = 20 μm. (I) Quantitative analysis of the mean fluorescence intensity of PINK1 and PARKIN in mice. (J, K) The m6A enrichment level of PINK1 was quantified by MeRIP-qPCR among the different experimental groups. (L) PINK1 mRNA levels were analyzed by RT-qPCR assay in OE-METTL3 cells after Actinomycin-D treatment for 0, 2, 4, and 6 (H) All data presented in this study are representative of at least three independent experiments. Data are presented as the mean ± SEM. PM2.5, particulate matter ≤2.5 μm.

Fig 4: METTL3 mediated PM2.5-induced mitophagy-dependent ferroptosis in bronchial epithelial cells. (A) H&E staining in lung sections; scale bars = 50 μm. (B) The inflammation scores of pulmonary airway. (C) Representative images and quantified mean fluorescence intensity of Acsl4 and xCt in mouse lung tissues from control and Re-Mettl3 groups; scale bars = 20 μm. (D) Representative images and quantified mean fluorescence intensity of Tom20 and Lc3b in mouse lung tissues from each group; arrows, co-localizations of Tom20 with Lc3b; scale bars = 20 μm. All data presented in this study are representative of at least three independent experiments. Data are presented as the mean ± SEM. PM2.5, particulate matter ≤2.5 μm.

Fig 5: Schematic model. Under homeostatic conditions (left), the normal m6A modification of PINK1 mRNA promotes its degradation, thereby sustaining basal mitophagy levels. Upon PM2.5 exposure (right), on the one hand, upregulated METTL3 expression enhances m6A modification of PINK1 mRNA; this modification stabilizes the transcript and consequently increases PINK1 protein levels. On the other hand, METTL3 also increases PARKIN expression. PINK1 then recruits PARKIN, subsequently triggering mitophagy. Excessive mitophagy disrupts redox homeostasis, inducing lipid peroxidation, which is characterized by elevated ACSL4 expression and reduced xCT expression, and ultimately drives ferroptosis, a central pathogenic event in PM2.5-induced lung injury. m6A, N 6-methyladenosine; PM2.5, particulate matter ≤2.5 μm.