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 ) The lack of Myc mRNA downregulation after prolonged TNF stimulation in B6.Sst1S macrophages. BMDMs from B6 and B6.Sst1S were treated with 10 ng/mL TNF for 6, 12, and 24 hr. Expression of Myc was quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated B6 BMDMs. β-actin was used as the internal control. ( B ) Myc protein levels expressed by B6 and B6.Sst1S BMDMs during the course of stimulation with TNF(10 ng/mL) for 6 and 12 hr. (western blot). Average densitometric values from two independent experiments were included above the blot. ( C ) Myc inhibition restored the levels of Fth and Ftl proteins in TNF-stimulated B6.Sst1S macrophages to the B6 levels. B6 and B6.Sst1S BMDMs were treated with 10 ng/mL TNF alone or in combination with Myc inhibitor, 10058-F4 (10 μM) for 24 hr. 10058-F4 was added 2 hr post TNF stimulation. Protein levels of Fth and Ftl were observed using western blot. Average densitometric values from two independent experiments were included above the blot. ( D ) Myc inhibition decreased the labile iron pool in TNF-stimulated B6.Sst1S macrophages. B6.Sst1S BMDMs were treated with 10 ng/mL TNF or left untreated for 48 hr. The 10058-F4 inhibitor was added 2 hr post TNF stimulation. The labile iron pool (LIP) was measured using the Calcein AM method and represented as fold change as compared to untreated control. DFO was used as a negative control, and FeSO4 was used as a positive control. ( E and F ) Myc inhibition reduced lipid peroxidation in TNF-stimulated B6.Sst1S BMDMs. Cells were treated with 10 ng/mL TNF in the presence or absence of 10058-F4 for 48 hr. The inhibitor was added 2 hr post TNF stimulation. The MDA production was measured using commercial MDA assay ( E ) The lipid peroxidation was measured by fluorometric method using C11-Bodipy 581/591 ( F ). ( G ) B6.Sst1S BMDMs were treated as above in E. The accumulation of lipid peroxidation product, 4-HNE after 48 hr was detected by confocal microscopy using 4-HNE-specific antibody. The 4-HNE adducts accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated group. ( H ) The BMDMs from B6.Sst1S were treated with 10 ng/mL TNF alone or in combination with Myc inhibitor, 10058-F4 (10 μM) for 24 hr. 10058-F4 was added 2 hr post TNF stimulation. Expression of Ifnb1 , Rsad2, Trib3, and Chac1 was quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated group. 18 S was used as the internal control. ( I and J ) B6 ( I ) and B6.Sst1S ( J ) BMDMs were treated with TNF (10 ng/ml) for 6, 12, and 24 hr in the presence or absence of JNK inhibitor D-JNK1 (2 μM). The cells were harvested and the protein levels of c-Myc and p-cJun were determined by western blotting. JNK inhibitor D-JNK1 was added 2 hr post TNF stimulation. Average densitometric values from two independent experiments were included above the blot. 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 Šídák’s multiple comparison test (Panel A) and ordinary one-way ANOVA using Šídák’s multiple comparison test (Panels D-F and H). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 5—source data 1. PDF file containing original western blots for indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in .
Article Snippet: Antibody , Rabbit polyclonal anti-phospho-cJun antibody , Cell Signaling Technology , Cat# 9261 s, RRID: AB_2130162 , WB (1:1000).
Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Inhibition, Negative Control, Positive Control, Multiple Displacement Amplification, Confocal Microscopy, Standard Deviation, Comparison
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