Journal: Clinical Science (London, England : 1979)

Article Title: Impaired cardiac non-neuronal acetylcholine synthesis triggers mitochondrial dysfunction with the loss of nicotinic receptor-mediated calcium handling, causing the failing heart

doi: 10.1042/CS20257026

Figure Lengend Snippet: A . LC3-II or LC3-I/II ratio was altered in hChAT KO mouse hearts, associated with the up-regulation of p62 and nitrotyrosine protein expression. B , Protein levels of both Parkin (PRK) and PINK1 were altered reciprocally between cytosolic and mitochondrial fractions of hChAT KO mouse hearts, compared with control mouse hearts. The hChAT KO reduced PRK and PINK1 protein levels in the cytosolic fraction ( P <0.05 and P <0.05, respectively), however alternatively, their levels both increased in the mitochondrial fractions ( P <0.01 and P <0.01, respectively).

Article Snippet: Furthermore, proteins from mitochondrial or cytosol fraction were subjected to western blot analysis using a rabbit anti-PARK2/Parkin polyclonal antibody (1:1000, #14060–1-AP; Proteintech), rabbit anti-PINK1 polyclonal antibody (1:1000, #23274–1-AP; Proteintech), rabbit anti-GAPDH monoclonal ab (Cell Signaling Technology), and rabbit anti-VDAC polyclonal ab (Abcam).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification orchestrates mitophagy-dependent ferroptosis in PM2.5-induced lung injury

doi: 10.3389/fimmu.2025.1683819

Figure Lengend Snippet: METTL3 drove PM2.5-induced mitophagy-dependent ferroptosis through m 6 A-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; m 6 A, N 6 -methyladenosine.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-FACL4 antibody (ab155282, Abcam, Cambridge, UK), rabbit anti-METTL3 antibody (ab195352, Abcam, UK), rabbit anti-xCT antibody (DF12509, Affinity, Shanghai, China), rabbit anti-PINK1 antibody (23274-1-AP, Proteintech, Wuhan, China), rabbit anti-PARK2 antibody (14060-1-AP, Proteintech, Wuhan, China), rabbit anti-TOM20 antibody (42406S, CST, Boston, USA), mouse anti-LC3B antibody (83506S, CST, Boston, USA), and mouse anti-β-actin antibody (66009-1-Ig, Proteintech, Wuhan, China), mouse anti- GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Western Blot, Quantitative RT-PCR, Staining

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification orchestrates mitophagy-dependent ferroptosis in PM2.5-induced lung injury

doi: 10.3389/fimmu.2025.1683819

Figure Lengend Snippet: METTL3 drove PM2.5-induced mitophagy-dependent ferroptosis through m 6 A-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 m 6 A 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.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-FACL4 antibody (ab155282, Abcam, Cambridge, UK), rabbit anti-METTL3 antibody (ab195352, Abcam, UK), rabbit anti-xCT antibody (DF12509, Affinity, Shanghai, China), rabbit anti-PINK1 antibody (23274-1-AP, Proteintech, Wuhan, China), rabbit anti-PARK2 antibody (14060-1-AP, Proteintech, Wuhan, China), rabbit anti-TOM20 antibody (42406S, CST, Boston, USA), mouse anti-LC3B antibody (83506S, CST, Boston, USA), and mouse anti-β-actin antibody (66009-1-Ig, Proteintech, Wuhan, China), mouse anti- GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Quantitative RT-PCR, Over Expression, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification orchestrates mitophagy-dependent ferroptosis in PM2.5-induced lung injury

doi: 10.3389/fimmu.2025.1683819

Figure Lengend Snippet: Mitophagy inhibition alleviated PM2.5-induced histological change in mice. (A) Lung tissues from different experimental groups were stained with hematoxylin and eosin (H&E); scale bars = 50 μm. (B) Representative images of periodic acid–Schiff (PAS) staining; scale bars = 50 μm. (C) The pulmonary inflammation score in each group. (D) The percentage of goblet cells in each group. (E) Total cells and neutrophils in BALF in different experimental groups. (F–I) The protein levels of Pink1, Parkin, Acsl4, and xCt in lung tissues in each group. 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; BALF, bronchoalveolar lavage fluid.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-FACL4 antibody (ab155282, Abcam, Cambridge, UK), rabbit anti-METTL3 antibody (ab195352, Abcam, UK), rabbit anti-xCT antibody (DF12509, Affinity, Shanghai, China), rabbit anti-PINK1 antibody (23274-1-AP, Proteintech, Wuhan, China), rabbit anti-PARK2 antibody (14060-1-AP, Proteintech, Wuhan, China), rabbit anti-TOM20 antibody (42406S, CST, Boston, USA), mouse anti-LC3B antibody (83506S, CST, Boston, USA), and mouse anti-β-actin antibody (66009-1-Ig, Proteintech, Wuhan, China), mouse anti- GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Inhibition, Staining

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification orchestrates mitophagy-dependent ferroptosis in PM2.5-induced lung injury

doi: 10.3389/fimmu.2025.1683819

Figure Lengend Snippet: Schematic model. Under homeostatic conditions (left), the normal m 6 A 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 m 6 A 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. m 6 A, N 6 -methyladenosine; PM2.5, particulate matter ≤2.5 μm.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-FACL4 antibody (ab155282, Abcam, Cambridge, UK), rabbit anti-METTL3 antibody (ab195352, Abcam, UK), rabbit anti-xCT antibody (DF12509, Affinity, Shanghai, China), rabbit anti-PINK1 antibody (23274-1-AP, Proteintech, Wuhan, China), rabbit anti-PARK2 antibody (14060-1-AP, Proteintech, Wuhan, China), rabbit anti-TOM20 antibody (42406S, CST, Boston, USA), mouse anti-LC3B antibody (83506S, CST, Boston, USA), and mouse anti-β-actin antibody (66009-1-Ig, Proteintech, Wuhan, China), mouse anti- GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Modification, Expressing

Journal: Journal of Inflammation Research

Article Title: IL-1β-Mediated Immunometabolic Adaptation in Corneal Epithelial Cells

doi: 10.2147/JIR.S495323

Figure Lengend Snippet: There was a decrease in mitophagy after prolonged exposure to IL-1β in corneal epithelial cells. ( A – C ) Immunoblotting was used to quantify mitophagy proteins, PINK1 and BNIP3L/NIX. Prolonged exposure to IL-1β showed a decrease in both proteins. ( B and C ) Corresponding densitometry values for ( B ) PINK1, and ( C ) BNIP3L/NIX. Proteins were normalized to vinculin, *P<0.05, n=3. ( D ) Transmission electron microscopy of cells treated with 100 ng/mL IL-1β for 72 hours showed the presence of intact cristae and mitochondrial hyperfusion (arrows). The dotted line in the top panels corresponds with the zoomed region in the bottom panels. Zoomed images show similar cristae morphology between treated and control cells. Scale bar: 1 μm (top panel); scale bar: 200 nm (bottom panel).

Article Snippet: The primary antibodies that were used included anti-rabbit PINK1, anti-rabbit BNIP3L/NIX, and anti-rabbit vinculin (Cell Signaling, Danvers, MA).

Techniques: Western Blot, Transmission Assay, Electron Microscopy, Control