Figure 5.

ROS scavenger NAC reduces BCG-induced macrophage ferroptosis. RAW264.7 macrophages were preincubated in medium containing 2.0 mM NAC for 1 h prior to being infected with BCG at an MOI of 5 for 24 h, before they were harvested for analysis. (A–D) The viability (A), intracellular Fe²⁺ (B), intracellular ROS (C), and lipid peroxidation (D) of RAW264.7 macrophages treated with the indicated conditions, as determined by Trypan Blue assay, iron ion probes, flow cytometry, and BODIPY 581/591 C11 assays, respectively. (E) Representative fluorescence images of BODIPY 581/591 C11-labeled lipoxidation of polyunsaturated fatty acids in RAW264.7 macrophages of the indicated conditions showed the reduction of BCG-induced lipoxidation in cells pretreated with NAC. Cell nuclei were counterstained with DAPI. (F) Representative blots and semi-quantitative analysis of Gpx4, Fsp1, and HO-1 proteins of RAW264.7 cells treated with the indicated conditions. The NAC pretreatment increased Gpx4 and Fsp1 expression, but decreased Hmox1 protein in BCG-infected cells. Data obtained from three independent experiments were processed using GraphPad Prism 8.0.1 software and ImageJ 1.52.a. All values are presented as mean ± SD (**p < 0.01, and ***p < 0.001; n = 3).