Journal: ACS Nano

Article Title: Microplastic-Induced Macrophage Dysfunction Drives Lung Tumor Progression through Glutathione Imbalance

doi: 10.1021/acsnano.5c15425

Figure Lengend Snippet: 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.

Article Snippet: Each membrane was incubated overnight at 4 °C with primary antibodies against TLR2 (Cell Signaling Technology, 13744S), TLR4 (Proteintech, 66350–1-Ig), TIRAP (Cell Signaling Technology, 13077), MyD88 (Cell Signaling Technology, 4283), TRAF6 (Cell Signaling Technology, 67591), P-JNK (Cell Signaling Technology, 9251), P-c-Jun (Cell Signaling Technology, 3270), NRF2 (Cell Signaling Technology, 12721), GCLM (Abcam, ab126704), SLC7A11 (Novus Biologicals, NB300–318), NQO1 (Santa Cruz Biotechnology, sc-32793), GCLC (Cell Signaling Technology, 52183), GPX1 (R&D systems, AF3798), GPX3 (R&D systems, AF4199), GPX4 (R&D systems, MAB5457), FTL (Santa Cruz Biotechnology, sc-390558;), FTH1 (Santa Cruz Biotechnology, sc-376594), and β-actin (Cell Signaling Technology, 8457).

Techniques: Gene Expression, RNA Sequencing, Quantitative Proteomics, Western Blot, Expressing, Two Tailed Test

Journal: JCI Insight

Article Title: IFN- γ is a therapeutic target in paraneoplastic cerebellar degeneration

doi: 10.1172/jci.insight.127001

Figure Lengend Snippet: (A) Tumor size, and (B) mouse weight loss of L7-HA-PCD mice treated with XMG1.2 (neutralizing anti–IFN-γ mAb) or control IgG1 from day 10 onward (n = 23 per group, 4 independent experiments; 2-way ANOVA post-hoc Sidak’s multiple comparison test, **P < 0.01). (C) Rotarod performance of L7-HA-PCD mice treated with XMG1.2 or control IgG1 (n = 16 mice per group, 3 independent experiments; Mann-Whitney U test, *P < 0.05; **P < 0.01). (D) Histological analysis at day 20 of the cerebellum of L7-HA-PCD mice treated with XMG1.2 or IgG1, from day 10 onward. Left: representative staining for calbindin (brown) and nuclear counterstaining with hematoxylin (blue). Scale bar: 100 μm. Right: quantitative assessment of Purkinje cell density in L7-HA-PCD mice treated with XMG1.2 or IgG1 (n = 23 mice per group from 4 independent experiments; Mann-Whitney U test, ***P < 0.01). (E) IFN-γ deficiency in Purkinje cell–specific CD8 T cells does not prevent the development of the PCD mouse model. Left, tumor size; middle, weight; and right, Purkinje cell density in L7-HA-PCD mice transferred with HA-specific CD4 T cells and either IFN-γ–sufficient or IFN-γ–deficient (CD8 IFN-γ–KO) HA-specific CD8 T cells; n = 10–11 mice per group, 2 independent experiments.

Article Snippet: The anti–IFN-γ mAb (XMG1.2; 100 μg/injection) (gift from NovImmune) was given every other day.

Techniques: MANN-WHITNEY, Staining

Journal: JCI Insight

Article Title: IFN- γ is a therapeutic target in paraneoplastic cerebellar degeneration

doi: 10.1172/jci.insight.127001

Figure Lengend Snippet: (A) Representative IHC for CD3+ cells (brown) with nuclear counterstaining with hematoxylin (blue) on cerebellar sections from L7-HA-PCD mice treated with XMG1.2 (neutralizing anti–IFN-γ mAb) or control IgG1. Scale bar: 100 μm. (B) Left: representative flow cytometry staining at day 16 for CD4 and CD8 gated on live cerebellum-infiltrating T cells (CD90.2+) from L7-HA-PCD mice treated with XMG1.2 or IgG1. Right: absolute numbers of CD4 and CD8 T cells infiltrating the cerebellum of L7-HA-PCD mice treated or not with XMG1.2 (n = 12 per group, 3 independent experiments; Mann-Whitney U test, **P < 0.01; ***P < 0.001). (C) Expression of the α4 integrin (CD49d) on CD4 and CD8 T cells from the cerebellum of L7-HA-PCD mice treated with XMG1.2 or IgG1 (n = 8 per group, 2 independent experiments; Mann-Whitney U test, *P < 0.05). Frequency of IFN-γ+ cells (D), proliferating (Ki67+) cells (E), and dead cells (F) among cerebellum-infiltrating CD4 and CD8 T cells of L7-HA-PCD mice treated with IgG1 or XMG1.2 (n = 8 per group, 2 independent experiments). Geomean of CD107a cells (G) and frequency of Granzyme B+CD8+ cells (H) among cerebellum-infiltrating T cells of L7-HA-PCD mice treated with IgG1 or XMG1.2 (n = 8 per group, 2 independent experiments).

Article Snippet: The anti–IFN-γ mAb (XMG1.2; 100 μg/injection) (gift from NovImmune) was given every other day.

Techniques: Flow Cytometry, Staining, MANN-WHITNEY, Expressing