Fig 2: Knockdown of Hmox1 increases BCG-induced macrophage ferroptosis. (A) Representative blots of Hmox1 protein in RAW264.7 cells transfected with siRNA to murine Hmox1 gene. siRNA was transfected by Lipofectamine™ RNAiMAX, and the protein was analyzed at 24 h post-transfection. The si-Hmox1 showed the most efficient knockdown of Hmox1 and was used in subsequent experiments. (B–E) Representative blots (B) and semi-quantitative analysis of Gpx4 (C), Fsp1 (D), Ncoa4 (E), and HO-1 (F) proteins of RAW264.7 cells treated with indicated conditions. The siRNA-mediated knockdown of Hmox1 amplified the inhibition of Gpx4 and Fsp1 expression in cells infected with BCG. (G–J) The viability (B), intracellular Fe2+ (C), intracellular ROS (D), and lipid peroxidation (E) of RAW264.7 macrophages treated with indicated conditions, as determined by Trypan Blue assay, iron ion probes, flow cytometry, and BODIPY 581/591 C11 assays, respectively. (K) Representative fluorescence images of BODIPY 581/591 C11-labeled lipoxidation of polyunsaturated fatty acids in RAW264.7 macrophages of the indicated conditions showed the increase of BCG-induced lipoxidation in cells transfected with siRNA to Hmox1. Cell nuclei were counterstained with DAPI. (L) Representative images of transmission electron microscopy showed mitochondrial membrane ridge breaks (arrows) in siRNA-transfected RAW264.7 and/or BCG-infected macrophages; the right panel shows the enlarged image of the boxed area in its corresponding image in the left panel. Data obtained from three independent experiments were processed using GraphPad Prism 8.0.1 software and ImageJ 1.52.a. One-way ANOVA was used to analyze the differences between groups. All values are presented as mean ± SD (**p < 0.01; ***p < 0.001; n = 3). Bars, 500 nm in the right panel and 200 nm in the left panel of K, and 25 µm in L. ns, no statistical difference.

Fig 3: Aortic HMOX1 mRNA and HO-1 protein expression, localization within the aortic wall, and bilirubin content in patients undergoing elective surgical repair or surgery because of rupture and AOD controls. A, Representative Western blot for HO-1 protein expression. The 2 marked HO-1 bands represent native (˜32 kDa) and a truncated (˜28 kDa) isoform of HO-1. B, Comparison of HMOX1 mRNA expression and (C) HO-1 protein expression in AOD, eAAA, and rAAA. Data are normalized to an internal control (=1). For HMOX1 mRNA expression in eAAA, aortic tissues from n=19 patients were available. This group included 2 to 3 different specimen from the same AAA. Because of this, the number of samples was increased to n=24. In rAAA, n=11 aortic specimen were available. This group included 2 different AAA samples from 3 patients and the number of analyzed samples increased to n=13. For HO-1 protein in eAAA, n=17 patients were included and 3 different AAA specimens from the same patient were analyzed. Because of this, the number of samples increased to n=20. In rAAA, n=9 patients were included and 2 different samples from the same AAA were analyzed in 2 patients. D and F, Separate quantification of the upper (˜32 kDa) and lower (˜28 kDa) HO-1 band. F, Representative slides for HO-1 immunohistochemistry in eAAA and rAAA specimen. Redstained areas represent positive HO-1 immunoreactivity. Cell nuclei (blue) were counterstained using Mayer's hemalum. The lumen (Lu), adventitia (Ad), and attaching thrombus (Th) are marked in red. G, Comparison of aortic bilirubin content in AOD, eAAA, and rAAA and (H and I) Spearman's correlation (r S) with HO-1 protein expression in eAAA and rAAA. In eAAA, pairs of bilirubin and HO-1 protein were available in n=11 patients only. B–E and G, All data are shown as scatter dot plots. The horizontal line depicts the median with range (B-E, G). The number of analyzed samples is given in the figures. Kruskal–Wallis and Dunn's multiple comparison test. *P<0.05 AOD vs eAAA. # P<0.05 AOD vs rAAA. Ad indicates adventitia; AOD, arterial occlusive disease; eAAA, electively treated AAA; HO-1, heme oxygenase-1; HMOX-1, heme oxygenase-1 gene; Lu, lumen; rAAA, ruptured AAA; and Th, thrombus.

Fig 4: 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).

Fig 5: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs(A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O2) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O2) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, a-tubulin served as a loading control.