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. 2020 Feb 25;9:e53627. doi: 10.7554/eLife.53627

Figure 5. The Trx1 system positively regulates the binding activity of NF-κB p65 to the DNA in BMDCs.

(A–E) Txnrd1fl/fl;Rosa26-CreERT2 mice and Txnrd1fl/fl littermates were injected with TAM to delete the Txnrd1 gene, and bone marrow cells were differentiated with GM-CSF to obtain BMDCs. (A) BMDCs were stimulated with LPS (400 ng/ml) for 10, 30, or 60 min and lysed for western blot. Expression of phospho-IκB-α, respective IκB-α, NF-κB p65, phospho-Erk1/2 and respective Erk1/2 was assessed with β-actin as a loading control. (B, C) WT or Txnrd1-deficient BMDCs were fixed at the indicated time points post LPS treatment, stained for DNA (DAPI), NF-κB p65 and actin (Phalloidin), and imaged using a DeltaVision system. Approximately 10 randomly chosen imaging fields encapturing a total of 50–100 nuclei were analyzed per sample per condition. (B) Nuclear and whole-cell masks were made using the DAPI and phalloidin channels, and NF-κB signal intensity within the masks was quantified. Shown is the nuclear NF-κB signal strength plotted as percentage of whole-cell NF-κB signal. (C) Depicted are example images of the samples of indicated times points post LPS treatment. In the merged images, DAPI and anti-NF-κB channels are shown in red and green, respectively. Scale bar represents 10 μm (top-left panel). Squares indicate fields, which are magnified in Figure 5—figure supplement 2. (D) WT or Txnrd1-deficient BMDCs were stimulated with LPS (400 ng/ml) for 100 min, and the recruitment of NF-κB p65 to the Il12b (top-left), Il1b (top-right), Il6 (bottom-left) and Nfkbia (bottom-right) promoters was assessed by p65 chromatin immunoprecipitation (ChIP) analysis and quantified by RT-PCR. ‘Promoter’ indicates the utilization of a primer pairs that amplify a fragment close to the NF-κB binding sites at the promoter region of the indicated genes, whereas ‘Ctr primers’ indicate primer pairs that were used as a control to amplify a region several kilobases away from the NF-κB binding sites (n = 2). (E) The NF-κB p65 binding activity to its DNA response element in nuclear extracts from BMDCs stimulated with LPS (400 ng/ml) for 40 min was assessed by an ELISA-based method (n = 3). Bar graphs and dot plots represent mean + standard deviation. Data are representative of two (A–D) or three (E) independent experiments. For each panel, a representative experiment with technical replicates is shown (D, E). Two-way ANOVA adjusted by Bonferroni's multiple comparison test was used in B: ns, not significant. One-way ANOVA adjusted by Tukey’s multiple comparison test was used in D, E: *p≤0.0332; **p≤0.0021; ***p≤0.0002; ****p≤0.0001.

Figure 5.

Figure 5—figure supplement 1. Txnrd1 deficiency does not affect TLR signaling.

Figure 5—figure supplement 1.

Txnrd1fl/fl;Rosa26-CreERT2 mice and Txnrd1fl/fl littermates were injected with TAM to delete the Txnrd1 gene, and bone marrow cells were differentiated with GM-CSF to obtain BMDCs. BMDCs were then stimulated with R837 (5 μg/ml) for 10, 30, or 60 min and lysed for western blot. Expression of phospho-IκB-α, respective IκB-α, NF-κB p65, phospho-Erk1/2 and respective Erk1/2 was assessed with β-actin as a loading control. Data are representative of two independent experiments.
Figure 5—figure supplement 2. Magnification of the microscopy images of Figure 5C.

Figure 5—figure supplement 2.

The highlighted areas in anti-NF-κB channels in Figure 5C are shown here in higher magnification.
Figure 5—figure supplement 3. Antioxidant supplementation does not rescue IL-12p40 cytokine production in Txnrd1-deficient BMDCs.

Figure 5—figure supplement 3.

Txnrd1fl/fl;Rosa26-CreERT2 mice and Txnrd1fl/fl littermates were injected with TAM to delete the Txnrd1 gene, and bone marrow cells were differentiated with GM-CSF to obtain BMDCs. (A) Quantification of ROS levels in unstimulated BMDCs as control or after 4 hr of stimulation with LPS (400 ng/ml) stained with CM-H2DCFDA via flow cytometry (n = 2 for the untreated group and n = 4 for the LPS-treated group). (B, C) BMDCs were then treated overnight with the antioxidants N-acetyl-L-cysteine (NAC; 0.9 mM), ascorbic acid (AA; 5 μM), DL-dithiothreitol (DTT; 1000 μM), diphenyleneiodonium chloride (DPI; 62.5 nM) (B) or catalase-polyethylene glycol (Cat; 250 U/ml) (C) before stimulation with R837 (5 ng/ml) for 7 hr. The concentration of IL-12p40 in supernatants was subsequently determined by ELISA (n = 3). (D) BMDCs were treated overnight with the antioxidant catalase-polyethylene glycol (Cat) before stimulation with LPS (400 ng/ml) for 3 hr. Shown are the ROS levels measured by staining with CM-H2DCFDA (n = 3). Bar graphs represent mean + standard deviation. Data are representative two independent experiments. For each panel, a representative experiment with replicates of in vitro culture conditions is shown. Student's t test (two-tailed, unpaired) was used for two-groups analysis (A–D): *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.