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: PM2.5 exposure promoted mitophagy-dependent ferroptosis in bronchial epithelial cells. (A) Scatter plots showing the correlation between mitophagy and ferroptosis in Beas-2B cells followed by PM2.5 exposure. (B, C) Mtphagy Dye and Lyso Dye staining for detection of mitophagy. (D) Immunofluorescence staining was performed to examine the expression of TOM20 and LC3B in cell models; arrows, co-localizations of TOM20 with LC3B; scale bars = 10 μm. (E) Immunofluorescence staining was performed to examine the expression of Tom20 and Lc3b in mouse models; arrows, co-localizations of Tom20 with Lc3b; scale bars = 20 μm. (F) MTT measured the cell viability of Beas-2B cells pretreated with different doses of Mdivi-1 for 2 h, followed by PM2.5 exposure. (G) Western blotting analysis of ACSL4 and xCT. (H, I) Liperfluo staining for the determination of lipid peroxidation; scale bars = 10 μm. (J, K) FerroOrange staining for detection of ferrous ions; 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.

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: Staining, Immunofluorescence, Expressing, Western Blot

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 mediated PM2.5-induced mitophagy-dependent ferroptosis in bronchial epithelial cells. (A, C) Representative immunofluorescence images of Mtphagy Dye and Lyso Dye; scale bars = 10 μm. (B, D) Representative immunofluorescent images of TOM20 and LC3B; arrows, co-localizations of TOM20 with LC3B; scale bars = 10 μm. (E, G) Representative immunofluorescence images of lipid peroxides; scale bars = 10 μm. (F, H) Representative immunofluorescence images of ferrous ions; scale bars = 10 μm. (I) Representative immunoblots and quantitative histogram of ACSL4 and xCT. (J) The cell viability was measured using MTT. 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: Immunofluorescence, Western Blot

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 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.

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: Staining, Fluorescence, Control

Journal: Nature Communications

Article Title: Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis

doi: 10.1038/s41467-022-30490-6

Figure Lengend Snippet: a – c RT-qPCR analysis of si CTRL and si ATG5 LEC treated with sodium acetate (AC, 20 mM, 48 h) or vehicle. mRNA expression of Prox1, VEGFR3, and LYVE1 (relative to HPRT). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 and *** p < 0.001 vs si CTRL, ## p < 0.01 and ### p < 0.001 vs si ATG5. d Representative blots for the indicated proteins in si CTRL or si ATG5 LEC treated with AC or vehicle. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05,*** p < 0.001. e Representative blots for indicated proteins in si CTRL and si ATG5 LEC treated with palmitate (PAL, 500 nM, 48 h) or BSA. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. f Representative immunofluorescent images of si CTRL and si ATG5 treated with AC or vehicle and stained for mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. g Quantification of mitochondrial fragmentation index (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05 vs si CTRL, # p < 0.05 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 12 cells analyzed per condition per independent experiment. h Quantification of lipid droplet number per cell. Mean ± SD, N = 4 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 29 cells analyzed per condition per independent experiment. i Representative super resolution AiryScan images of si CTRL and si ATG5, si CTRL + si DRP1, and si ATG5 + si DRP1 LEC stained for the mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. j Quantification of mitochondrial index of fragmentation (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, **** p < 0.0001 vs si CTRL, #### p < 0.0001 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 35 cells analyzed per condition per independent experiment. k Quantification of lipid droplet number per cell. Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 47 cells analyzed per condition per independent experiment. l – o RT-qPCR analysis in si CTRL, si ATG5, si CTRL + si DRP1 and si ATG5 + si DRP1 LEC. mRNA expression of PROX1, VEGFR3, LYVE1, and CPT1A (relative to HPRT). Mean ± SD, N = 3 ( l , n , o ) and N = 4 ( m ) biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 vs si CTRL and p = ns vs si ATG5.

Article Snippet: Primary antibodies used were rabbit anti-ATG5 (12994S, CST), rabbit anti-ATG7 (1:1000, 8558S, CST), rabbit anti-LC3 (1:1000, 3868S, CST), rabbit anti-GAPDH (1:5000, 2118S, CST), rabbit anti-p62 (1:1000, p0067, Millipore), rabbit anti-β-actin (1:5000, A5441, Sigma-Aldrich), goat anti-LYVE1 (1:1000, AF2089, R&D systems), rabbit anti-PROX1 (1:1000, 11067-2, Proteintech), rabbit anti-VEGFR3 (1:1000, ab154079, Abcam), mouse anti-NR2F2 (1:1000, ab41859, Abcam), rabbit anti-CPT1 (1:1000, D3B3, CST) antibody, rabbit anti-CPT2 (1:1000, ab18114, Abcam), rabbit anti-acetyl histone H3 (lysine 9) antibody (1:1000, 9671, CST), rabbit anti-pan-acetyl histone H3 antibody (1:1000, 39139, Active Motif)), mouse anti-CD36 antibody (1:1000; ab17044, Abcam), mouse anti-ULK1 antibody (1:1000; ab56344, Abcam), rabbit anti-TOMM20 (1:1000; BD612278, BD Biosciences), mouse anti-OPA1 (1:1000; 612607, BD Biosciences), mouse anti-ENOS (1:1000; 610297, BD biosciences), rabbit anti-phospho DRP1 (Ser616) (1: 1000; 3455S, CST), mouse anti-DRP1 (1:1000; 611113, BD Biosciences), rabbit anti-H3K4me antibody (1:1000; ab8580, Abcam), rabbit anti-H3K27 ac (1:1000; 8173T, CST) and rabbit anti-FABP4 antibody (1:1000; 2120S, CST).

Techniques: Quantitative RT-PCR, Expressing, Staining

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Schematic of Raf1 BioID and localization workflow. ( B ) SAINT plots demonstrating all hits with a positive SAINT score for proteins enriched in the proximal proteome of Raf1 in dependent MM485 or AsPC1 cells compared to Raf1-independent CHL1 and BxPC3 and eGFP controls. Red are select known Raf1 interactors and cyan indicates proteins of interest involved in metabolic processes. Dashed line drawn at SAINT score of 0.8 ( C ) Combined Cell Component, Biological Process, and Molecular Function Gene Ontology Enrichment in SAINT ≥ 0.8 proteins. Benjamini-Hochberg adjusted pValues are used. ( D ) Network of Raf1 proximal proteins with SAINT ≥ 0.9 in both pancreatic and melanoma cell lines of interest. Edges between non-Raf1 proteins represent known interactions. Proteins with known mitochondrial localization labeled with blue. ( E ) Mito-tag Mitochondrial isolation of MM485 melanoma cells. WCL denotes whole cell lysate, Mito indicates mitochondrial fraction, and PK denotes proteinase K treatment to digest outer mitochondrial membrane (OMM) proteins. Lamin A/C is a nuclear (nuc) protein, p70S6 kinase is a cytoplasmic (cyto) protein, and TOMM20 spans the mitochondrial outer mitochondrial membrane (OMM) ( F ) STED microscopy demonstrating inner-mitochondrial Raf1 (yellow arrows). Image stack side-view (XZ and YZ) are cropped laterally and then mean-projected along X or Y. Inset displays a Y-projection of a particular mitochondria of interest in XZ.

Article Snippet: Two-color confocal and 3D STED imaging of primary Mouse Raf1 (12552, Cell Signaling Technology) with Abberior STAR Red antiMouse and rabbit TOMM20 (ab78547) with antiAbberior STAR Orange was performed on an Abberior Facility Line STED microscope.

Techniques: Labeling, Isolation, Membrane, Microscopy