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

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© 2020, Muri et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 4. — (A–J) Txnrd1^fl/fl;Rosa26-CreERT2 mice and Txnrd1^fl/fl littermates were injected with TAM to delete the Txnrd1 gene. Bone marrow cells were differentiated with GM-CSF to obtain BMDCs. (A) Analysis of Txnrd1 mRNA by RT-PCR in cultured Txnrd1^fl/fl;Rosa26-CreERT2 and Txnrd1^fl/fl BMDCs for confirmation of gene deletion (n = 3). (B) Viability (eFluor780^-Annexin-V^-) of BMDCs was assessed via flow cytometry (n = 3). (C) BMDCs were primed with CpG (100 nM), LPS (100 ng/ml), LTA (1 μg/ml), R837 (5 μg/ml) for 7 hr, and expression of Txn1 at the mRNA level was determined via RT-PCR (n = 3). (D, E) BMDCs were stimulated for 7 hr with CpG (100, 20 nM), LPS (100, 20 ng/ml), LTA (5, 1 μg/ml), R837 (5, 1 μg/ml), or zymosan (10, 2 μg/ml). ‘High’ and ‘Low’ indicate the concentration of the utilized stimulus. IL-6 (D) and IL-12p40 (E) were measured in supernatants by ELISA (n = 3). (F, G) BMDCs were stimulated for 7 hr with CpG (100 nM), LPS (100 ng/ml), LTA (1 μg/ml) or R837 (5 μg/ml), and the expression of Il6 (F) and Il12b (G) was determined by RT-PCR (n = 3). (H) BMDCs were stimulated with CpG (100 nM), LPS (400 ng/ml), LTA (5 μg/ml), R837 (5 μg/ml) or zymosan (10 μg/ml) before the addition of 2 mM ATP or 200 μg/ml alum for 1 hr or 4 hr, respectively. The concentration of IL-1β in supernatants was determined by ELISA (n = 3). (I) BMDCs were stimulated with TLR ligands as in F,G), and expression of Il1b at the mRNA level was determined via RT-PCR (n = 3). (J) Naive, splenic, Smarta-1 transgenic CD4⁺ T cells were co-cultured with Txnrd1-deficient BMDCs (or Txnrd1-sufficient BMDCs as a control) and the indicated concentrations of the GP_61-80 peptide. Shown are the frequencies of CD4⁺ T cells producing IFN-γ⁺ after restimulation with PMA/ionomycin (n = 3). Bar graphs represent mean + standard deviation. Data are representative of two (A–C, F, G, I, J) or four (D, E, H) 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 to compare Txnrd1^fl/fl;Rosa26-CreERT2 and control Txnrd1^fl/fl groups in (A, B, D–J): *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns, not significant. One-way ANOVA followed by Dunnett’s correction was used in C (comparison to the unstimulated control): *p≤0.0332; **p≤0.0021.

Figure 4—figure supplement 1. — (A–J) Txnrd1^fl/fl;Rosa26-CreERT2 mice and Txnrd1^fl/fl littermates were injected with TAM to delete the Txnrd1 gene. Bone marrow cells were differentiated with GM-CSF to obtain BMDCs. (A) Analysis of Txnrd1 mRNA by RT-PCR in cultured Txnrd1^fl/fl;Rosa26-CreERT2 and Txnrd1^fl/fl BMDCs for confirmation of gene deletion (n = 3). (B) Viability (eFluor780^-Annexin-V^-) of BMDCs was assessed via flow cytometry (n = 3). (C) BMDCs were primed with CpG (100 nM), LPS (100 ng/ml), LTA (1 μg/ml), R837 (5 μg/ml) for 7 hr, and expression of Txn1 at the mRNA level was determined via RT-PCR (n = 3). (D, E) BMDCs were stimulated for 7 hr with CpG (100, 20 nM), LPS (100, 20 ng/ml), LTA (5, 1 μg/ml), R837 (5, 1 μg/ml), or zymosan (10, 2 μg/ml). ‘High’ and ‘Low’ indicate the concentration of the utilized stimulus. IL-6 (D) and IL-12p40 (E) were measured in supernatants by ELISA (n = 3). (F, G) BMDCs were stimulated for 7 hr with CpG (100 nM), LPS (100 ng/ml), LTA (1 μg/ml) or R837 (5 μg/ml), and the expression of Il6 (F) and Il12b (G) was determined by RT-PCR (n = 3). (H) BMDCs were stimulated with CpG (100 nM), LPS (400 ng/ml), LTA (5 μg/ml), R837 (5 μg/ml) or zymosan (10 μg/ml) before the addition of 2 mM ATP or 200 μg/ml alum for 1 hr or 4 hr, respectively. The concentration of IL-1β in supernatants was determined by ELISA (n = 3). (I) BMDCs were stimulated with TLR ligands as in F,G), and expression of Il1b at the mRNA level was determined via RT-PCR (n = 3). (J) Naive, splenic, Smarta-1 transgenic CD4⁺ T cells were co-cultured with Txnrd1-deficient BMDCs (or Txnrd1-sufficient BMDCs as a control) and the indicated concentrations of the GP_61-80 peptide. Shown are the frequencies of CD4⁺ T cells producing IFN-γ⁺ after restimulation with PMA/ionomycin (n = 3). Bar graphs represent mean + standard deviation. Data are representative of two (A–C, F, G, I, J) or four (D, E, H) 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 to compare Txnrd1^fl/fl;Rosa26-CreERT2 and control Txnrd1^fl/fl groups in (A, B, D–J): *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns, not significant. One-way ANOVA followed by Dunnett’s correction was used in C (comparison to the unstimulated control): *p≤0.0332; **p≤0.0021.