Fig. 2

LCN2-mediated ROS production is involved in MHC class I molecule expression during Mtb infection. A BMDCs derived from WT and LCN2^−/− mice were infected with Mtb at an MOI of 1 for the indicated time periods, then stained using ROS-ID Total ROS detection kit (1 µM, 37 °C, 30 min). Data are shown as mean ± SD (n = 12). B BMDCs derived from WT and LCN2^−/− mice were stimulated with LPS (100 ng/ml) for 24 h, then stained using ROS-ID Total ROS detection kit (1 µM, 37 °C, 30 min). Data are shown as mean ± SD (n = 5). Intracellular ROS level was detected by flow cytometry in CD11c⁺-gated BMDCs. C, D BMDCs derived from WT and LCN2^−/− mice were infected with Mtb at an MOI of 1 for the indicated time periods. Western blotting was performed using antibodies against PDI, ERO1α, p47phox, NRF2 and HO-1. LPS (1 µg/ml, 1 h) were used as positive controls. Western blot bands corresponding to each protein were quantified, and the intensity of each target protein was normalized to the intensity of the β-actin loading control. The normalized ratio of the WT unstimulated control (UN) was set as 1.0 to compare target protein abundance in different samples. The normalized ratio is shown at the bottom of the blots. A representative blot from three independent experiments is shown. E BMDCs derived from WT and LCN2^−/− mice were preincubated with NAC (0.2 or 0.5 mM) or DPI (0.5 µM) for 30 min, then infected with Mtb at an MOI of 1 for 48 h. The expression of MHC class I molecules was analyzed by flow cytometry on CD11c⁺‐gated BMDCs. Data are shown as mean ± SD (n = 6). Statistically significant differences were determined by Mann–Whitney test (non‐parametric unpaired two-tailed t-test; *) or Wilcoxon signed-rank test (non‐parametric paired two-tailed t-test; ^#). *p < 0.05, **p < 0.01, and ***p < 0.001; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001