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: Journal of Biological Chemistry

Article Title: Developmental Control of Apoptosis by the Immunophilin Aryl Hydrocarbon Receptor-interacting Protein (AIP) Involves Mitochondrial Import of the Survivin Protein

doi: 10.1074/jbc.m110.210120

Figure Lengend Snippet: FIGURE 6. Tom20 regulation of mitochondrial import of survivin. A, ali- quots of GST, GST-Tom20, or GST-Tom70 recombinant proteins were mixed with Raji cell extracts, and bead-bound material was analyzed by Western blotting. Bottom panel, Coomassie Blue staining of recombinant GST fusion proteins. B, Raji mitochondrial extract was immunoprecipitated with an anti- body to survivin or IgG, and proteins in pellets or supernatants (unbound) were analyzed by Western blotting. C, recombinant wild-type survivin or the survivin 1–141 mutant and GST-Tom20 beads were mixed with increasing concentration of recombinant AIP, and bead-bound proteins were analyzed by Western blotting (top panel). Bottom panel, GST or GST-Tom20 was incu- bated with recombinant AIP in the presence of increasing concentrations of survivin (up to 0.4 M), and bound proteins were analyzed by Western blot- ting. D, independently established clones (#) of HeLa cells stably transfected with control (Ctrl) or Tom70- or Tom20-directed shRNA were analyzed by Western blotting. E, HeLa cells were transfected with control, non-targeting (Ctrl) or Tom20-directed siRNA and analyzed by Western blotting. TCE, total cell extracts. F, clones of HeLa cells with stable knockdown of Tom70 or Tom20 were treated with 0.5 M staurosporine for 24 h and analyzed for cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. DataaremeanS.E.ofreplicates(n3).***,p0.0001.Twodifferentclones per conditions were tested with identical results in two independent experiments.

Article Snippet: Antibodies against AIP (Novus Biologicals), survivin (Novus Biologicals), Tom20 (Santa Cruz Biotechnology), Tom70 (Novus Biologicals), COX-IV (Clontech), Smac (Pro-Sci), -actin (Sigma), and Hsp90 (BD Biosciences) were used.

Techniques: Recombinant, Western Blot, Staining, Immunoprecipitation, Mutagenesis, Concentration Assay, Clone Assay, Stable Transfection, Transfection, Control, shRNA, Knockdown

Journal: Cells

Article Title: Korean Red Ginseng-Induced SIRT3 Promotes the Tom22-HIF-1α Circuit in Normoxic Astrocytes.

doi: 10.3390/cells12111512

Figure Lengend Snippet: Figure 5. HIF-1α-mediated expression of mitochondria, Tom20, and Tom22 in KRGE-treated in vitro astrocytes. (a,b) Human brain astrocytes were transfected with indicated si-RNA and added with KRGE (0.5 mg/mL) for 24 h. * p < 0.05; ** p < 0.01; *** p < 0.001. (a) Protein levels were analyzed using western blot and quantified (n = 5–6). (i) water + negative control si-RNA; (ii) KRGE + negative control si-RNA; (iii) KRGE + si-ERRα; (iv) KRGE + si-HIF-1α; (v) KRGE + si-ERRα + si-HIF-1α. (b) Protein levels were determined using western blot and quantified (n = 3–6 independent cell cultures).

Article Snippet: At 70~75% confluency, astrocytes were transfected with small interfering ribonucleic acid (si-RNA) against a negative control (Thermo Fisher Scientific) or SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, nicotinamide phosphoribosyltransferase (Nampt), ERRα, Tom20, Tom22, PHD2 (Santa Cruz Biotechnology), and HIF-1α (Dharmacon, Lafayette, CO, USA) using RNAiMax (Thermo Fisher Scientific).

Techniques: Expressing, In Vitro, Transfection, Western Blot, Negative Control