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. 2019 Sep 18;10:2147. doi: 10.3389/fimmu.2019.02147

Figure 7.

Figure 7

AMPK and HECT E3 ligase are required for Txnip degradation in TLR2-mediated inflammation. RAW264.7 cells were pretreated with (A,B) heclin (25 μM) or (C,D) dorsomorphin (10 μM) for 1 h followed by stimulation with HK-GAS (MOI 10), LTA (5 μg/ml), and PGN (5 μg/ml). The expression of Txnip and GAPDH was detected at 2 h by Western blotting. The expression ratios of Txnip to GAPDH are shown. Protein molecular weights (MW) are indicated in kilodaltons. Western blot results represent at least two independent experiments. ELISA showed IL-6 and TNF-α production at 24 h in HK-GAS-, LTA-, and PGN-stimulated cells, and the concentrations were shown as the means ± SD of triplicate cultures. **p < 0.01 and ***p < 0.001 compared to the untreated group; #p < 0.05 and ###p < 0.001 compared to each stimulated group. (E) In the presence of heclin and dorsomorphin, the Griess reaction showed NO generation in HK-GAS-, LTA-, and PGN-stimulated cells at 24 h. The measurements are shown as the means ± SD of triplicate cultures. ***p < 0.001 compared to the untreated group; ###p < 0.001 compared to each stimulated group. (F) A represented model of that GAS infection induces the TLR2/NOX2-dependent rapid degradation of Txnip via AMPK- and HECT E3-ligase-regulation, which subsequently potentiates downstream inflammatory TNF-α, IL-6, and iNOS/NO generation.