Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) Total levels of Nrf1, β-TrCP, and Keap1 proteins in B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 8, 12, and 24 hr (western blotting). The experiment was repeated two times and shown the representative blot. ( B ) B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 12 hr. Total NRF1 levels were evaluated using confocal microscopy. Scale bar = 20 μm. ( C ) B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 12 hr. Nrf2 nuclear translocation was quantified using automated microscopy (Operetta CLS High Content Analysis System). Untreated samples were considered 100%. ( D ) The total antioxidant capacity of B6 and B6.Sst1S BMDMs was measured after TNF (10 ng/mL) stimulation. The percentage of induced antioxidant capacity upon TNF stimulation was plotted (Y axis). ( E and F ) B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 8 and 12 hr. Hmox1 and Nqo1 expressions were quantified using qRT-PCR. ( G ) The heatmap of all genes related to response to oxidative stress (gene ontology category GO: 0006979). The heatmap was generated using FPKM values obtained using bulk RNAseq of B6.Sst1S and B6 BMDMs after 12 hr of TNF stimulation. For heatmap generation, FPKM values were scaled using Z-scores for each tested gene. The data are presented as means ± standard deviation (SD) from three to five samples per experiment, representative of three independent experiments. The statistical significance was performed by two-way ANOVA using Tukey’s multiple comparison test (Panels C-F). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Rabbit polyclonal anti-KEAP1 antibody (reactive to human, mouse, rat) , Proteintech Group Inc , Cat# 10503–2-AP, RRID: AB_2132625 , WB (1:5000).

Techniques: Western Blot, Confocal Microscopy, Translocation Assay, Microscopy, High Content Screening, Quantitative RT-PCR, Generated, Standard Deviation, Comparison