Fig. 3
From: Involvement of cigarette smoke-induced epithelial cell ferroptosis in COPD pathogenesis
CS induces ferroptosis in mouse models. a, c, d WT mice were exposed to Room Air (RA) or cigarette smoke (CS) for 6 months. Ferric iron deposits were stained with Perls’ DAB staining in lung samples from RA (upper) or CS (lower) exposed mice. Counter staining is Fast red. Image (original magnification, ×400) is representative of 5 images/mouse. 3 biologically independent mice/group were analyzed. Scale bar = 100 µm. b The amount of non-heme iron was measured by means of inductively coupled plasma mass spectrometry (ICP-MS) (n = 4 biologically independent mice). *P < 0.05 by student’s t-test. c WB showing expression levels of ferritin and β-actin in lung homogenate of WT mice. *P < 0.05 by student’s t-test. (n = 7 biologically independent mice). d WB showing expression levels of NCOA4 and β-actin in lung homogenates from RA (n = 4) or CS (n = 4) exposed mice. (n = biologically independent mice.) *P < 0.05 by student’s t-test. e–j Heterozygous GPx4-deficient mice (GPx4+/−), GPx4+/+ (WT), and TG(loxP-GPx4):GPx4+/+ (GPx4TG) mice were exposed to RA or CS for 4 weeks. e LC-MS analysis of PC and their oxidation products in lung homogenates. PC-OOH/PC (16:0/18:2), (16:0/20:4) were shown. f LC-MS analysis of PE and their oxidation products in lung homogenates. PE-OOH/PE (16:0/18:2), (16:0/20:4) were shown. e, f TG RA n = 5,TG CS n = 6, WT RA n = 4,WT CS n = 4, +/− RA n = 4, +/− CS n = 6. (n = biologically independent mice) *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. g Immunohistochemical(IHC) staining of 4-HNE in mouse lung airway (upper panels) and parenchyma (lower panels). Original magnification ×400. Bar = 100 µm. h TUNEL assay staining (green) in GPx4 +/−, WT, and GPx4 TG mice lung sections. Nuclei were counterstained with DAPI (blue). TG RA n = 3,TG CS n = 4, WT RA n = 3,WT CS n = 3, +/− RA n = 3, +/− CS n = 5. (n = biologically independent mice)**P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. i Immunofluorescence staining of cleaved caspase3 (red). Nuclei were counterstained with DAPI (blue). Original magnification ×200. Bar = 100 µm. j WB showing expression levels of caspase 3, cleaved caspase3, and β-actin in lung homogenate of GPx4+/−, WT, and GPx4 TG mice. All bar charts shown represent the mean ± SEM
