Fig. 7: METTL3/YTHDF3-m6A/PTX3/STX17 axis controls autophagy maturation in macrophages. | Nature Communications

Fig. 7: METTL3/YTHDF3-m6A/PTX3/STX17 axis controls autophagy maturation in macrophages.

From: RNA m6A methylation modulates airway inflammation in allergic asthma via PTX3-dependent macrophage homeostasis

Fig. 7

a Human THP1-derived macrophages were infected with PTX3 siRNA pools (200 nM) or control, followed by IL-4 stimulation. KEGG analysis of pathways enriched in PTX3-deficient macrophages. b Transmission electron microscopy (TEM) determined the number of autophagosomes in control and PTX3-knockdown BMDMs (n = 4 cells). Representative images were shown together with mean values of the number of autophagosomes per cell. Scale bars, 1 μm. c Control and PTX3-deficient BMDMs were transiently transfected with mRFP-GFP-LC3-expressing adenovirus, followed by autophagy induction. The LC3 puncta were analyzed by confocal microscopy, and the mean number of LC3 puncta (top) was determined by counting (n = 4 cells). Scale bars: 10 μm. TEM demonstrates the decreased autophagosomes in Mettl3 KO BMDMs (d) (n = 4 animals) and YTHDF3-deficient THP1-derived macrophages (e) (n = 4 cells). Scale bars, 2 μm, and 1 μm, respectively. f, g The autophagy flux analysis showing the number of LC3 puncta in Mettl3 KO BMDMs (n = 4 animals) and YTHDF3-deficient THP1-derived macrophages (n = 4 cells). Scale bars, 25 μm, and 10 μm, respectively. h Heatmap identifying the down-regulated transcripts in PTX3-deficient THP1-derived macrophages. i RT-qPCR (n = 4 cells) and immunoblot (n = 3 cells) verifying reduced levels of STX17 in PTX3-knockdown BMDMs. j RT-qPCR showing up-regulated STX17 expression in Mettl3 KO BMDMs (n = 4 animals for WT group and n = 3 animals for KO group) and YTHDF3-deficient THP1-derived macrophages (n = 5 cells for control group and n = 4 cells for knockdown group), respectively. k The increased STX17 levels in Mettl3 KO BMDMs (n = 3 animals) and YTHDF3-deficient THP1-derived macrophages (n = 3 cells) by Western blot. l Immunoblot analysis of LC3, STX17, and PTX3 levels in control and PTX3-knockdown BMDMs treated with rapamycin for 0–6 h (n = 3 cells). Statistical analysis of the data was performed using two-sided unpaired t test with Welch’s correction (i left) or not (a, b, d, e, g, i right, j, k), and 2-way ANOVA (l) followed by Sidak’s multiple comparison tests. Data are presented as means ± SEM from one of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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