Fig 2: METTL14 regulates autophagy and biological functions of OSCC cells by mediating eIF4G1 expression. Transwell assays were used to detect the migratory and invasive abilities of HSC3 and CAL33 cells (one-way ANOVA). Proliferation of HSC3 and CAL33 cells was detected using CCK8 assays (one-way ANOVA). One group of cells were transfected with OEMETTL14 or OECtrl and cultured with or without 3MA (A–C), while the other group was silenced using siMETTL14 or siCtrl and rescued using rapamycin or not (D–F). (G) Western blot was used to compare expression levels of eIF4G1, LC3-I, and LC3-II in HSC3 and CAL33 cells transfected with OECtrl, OEMETTL14, and OEMETTL14+OEeIF4G1. (H–J) The migration, invasion, and proliferation of OSCC cells with eIF4G1 overexpression and METTL14 knockdown. (p or adjusted-p *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001).

Fig 3: Necroptotic Mϕ induce inflammatory responses in naive Mϕ. mRNA expression of IL1β, IL-6, IL-12, TNF-α, and NOS2 in naive BMDM placed in the bottom of a Transwell plate, with WT or LC3−/− BMDM pre-stimulated by BCM (40 μl/ml) for 24 h in the upper wells. Cells were then cocultured for 0–18 h. All results are representative of three independent experiments. The graphs show the mean and S.E.M., n=3. Significances between groups were determined by using independent samples two-tailed Student’s t-test. *P<0.05 and **P<0.01 compared with control groups

Fig 4: Fragmented mtDNA induces macrophage autophagy. (a) Western blot of whole-cell lysates showing LC3 expression in BMDM stimulated by 40 μl/ml BCM for 0–24 h. GAPDH used as loading control. (b) Western blot of whole-cell lysates showing LC3 expression in BMDM treated with 40 μl/ml BCM or 10 μg/ml CIRP for 24 h +/− IgG isotype antibody (10 μg/ml) or CIRP neutralizing antibody (10 μg/ml). (c) Western blot of whole-cell lysates showing LC3 expession in WT or gp91phox−/− BMDM stimulated by 40 μl/ml BCM or 10 μg/ml CIRP for 24 h. (d) Western blot of whole-cell lysates showing LC3 expression in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μl/ml) for 24 h. (e) Confocal immunofluorescence images and analysis showing colocalization of LC3 (autophagosome; green) and TUNEL (fragmented DNA; red) and Hoechst nuclear stain (blue) in BMDM treated with 40 μl/ml BCM or 10 μg/ml CIRP for 24 h. (f) Confocal immunofluorescence images and analysis of colocalization of LC3 (autophagosome; green) and TUNEL (fragmented DNA; red) and Hoechst nuclear stain (blue) in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μl/ml) for 24 h. (g) Confocal immunofluorescence images showing colocalization of mitochondria (MitoTracker; green) and lysosomes (LysoTracker; red) in BMDM, or J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μl/ml) for 24 h. (h) Expression levels of p62 mRNA or (i) protein expression in BMDM stimulated by BCM (40 μl/ml) for 0–24 h. (j) Western blot of whole-cell lysates showing p62 protein expression in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, then stimulated with BCM (40 μl/ml) for 0–6 h. (k) Confocal immunofluorescence images and analysis of colocalization of mitochondria (MitoTracker; green), LC3(autophagosomes; red) and p62 (cyan) in BMDM stimulated with BCM (40 μl/ml) or CIRP (10 μg/ml) for 24 h. Arrows indicate the colocalization of mitochondria-LC3-p62 (white). All results are representative of three independent experiments. The graphs show the mean and S.E.M., n=3. Significances between groups were determined by using independent samples two-tailed Student’s t-test. *P<0.05 and **P<0.01 versus the control or compared between the indicated groups

Fig 5: Psychosine and α-synuclein subcellular localization in vivo. Thioflavin-S (thio-S)-positive deposits in the P40 TWI brain co-localized with both the lysosomal marker Lamp-1 (A) and the autophagosomal marker LC3-I (B). TWI autophagosomes were enlarged compared to WT (C) and no aggregated thio-S material was detected in the WT brain (D). Anti-α-synuclein antibodies co-localized intracellularly with thio-S and the lysosomal marker LAMP-1 (E–I, arrows). Thio-S-positive α-synuclein was not detected in WT tissue (J). Isolation of lysosomes further confirmed α-synuclein co-localization with enriched lysosomes (K) and autophagosomes (L) while also staining positive for thio-S (K,L white arrows). Not all organelles that co-localized with α-synuclein were positive for thio-S (L, yellow arrows). Psychosine levels measured in total lysate and isolated lysosomes showed an accumulation in TWI that was significantly enriched in the lysosome/autophagosome fraction compared to the total lysate (n = 3, p < 0.05, ANOVA) (M). Fractions enriched in lysosomes and autophagosomes from TWI and WT brains showed an elevated level of Lamp-1 and LC3-I in addition to high-molecular-weight aggregates of α-synuclein in TWI (N). Ultrastructure EM imaging revealed fibrous material enveloped in apparent autophagic vacuoles (O, double membrane organelle marked by clack arrows), and immunoelectron micrography identified α-synuclein deposits within double-membrane (arrowheads, inset in Q) autophagosome structures (P,Q) in neurons from P40 TWI. Non-specific binding of secondary antibody (anti-IgG-Au, 0.8 nm) was not observed (R). Scale bars: 10 μm (A–L), 0.2 μm (O), 1 μm (P), and 100 nm (R). Uncropped blots displayed in Supplementary Fig. 18.