Fig 2: MP exposure impairs ROS detoxification in BMDMs through the dysregulation of the glutathione pathway. (A) Heatmap showing gene expression patterns with differential expressions. (B) Gene ontology (GO) enrichment analysis of DEGs in MP-treated BMDMs. Top stress-related pathways, including response to oxidative stress and detoxification, are ranked by the normalized enrichment score (NES). (C) ROS production in MP-treated BMDMs, quantified relative to CTL. (D) Nitrite production in MP-treated BMDMs, quantified relative to CTL. (E) Gene set enrichment analysis (GSEA) of the oxidative stress response gene set based on total RNA-seq data from MP-treated BMDMs compared with that from untreated CTL cells. The enrichment score curve and NES indicate substantial up-regulation of the oxidative stress response genes in MP-treated BMDMs. (F) Heatmap showing the differential expression of oxidative stress response-involved genes in MP-treated BMDMs compared with that of untreated CTL cells. Genes up- and down-regulated by MP exposure are marked in red and blue, respectively. The most substantially up- and down-regulated glutathione metabolism-related genes are highlighted. Glutathione metabolism-related genes, such as Gclm, Slc7a11, Nqo1, Gclc, Gpx1, and Gpx3, are highlighted. (G) Volcano plot depicting differential expressions of glutathione metabolism-related genes in MP-treated BMDMs compared with that in CTL cells. (H) Representative Western blot images showing the expression of NRF2 and that of its downstream antioxidant-related targets (GCLM, SLC7A11, NQO1, and GCLC) in BMDMs treated with MPs for 24 h. (I) Glutathione-dependent antioxidant enzyme GPX1 and GPX3 expression was analyzed using β-actin as a loading CTL. (J) GSH/GSSG ratio and total glutathione level quantifications in MP-treated BMDMs relative to the CTL cells. a.u., arbitrary unit. The data represent at least three independent experiments and are expressed as the mean ± SEM, using unpaired two-tailed t tests. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.