Fig 2: Long-term CS exposure led to emphysema and skeletal muscle dysfunction. (A–F) Airway resistance (RL), lung compliance, FVC, FEV0.1, FEV0.1/FVC, and FEF25-75% were measured in mice; (G) H&E staining of lung tissues from mice exposed to air and CS; Arrows (left) indicate destruction of the alveolar wall; Arrow (right) indicate Inflammatory cells infiltrates in the lung parenchyma and interstitial spaces; The scale in each image is 100 µm; (H and I) histogram of MLI and DI; (J) FACS showed the neutrophils (CD11B+Ly6G+) and eosinophils (CD11B+SiglecF+) in BALF; (K) histogram of grip strength; (L) H&E staining of muscles from mice exposed to air and CS; Arrows indicate changes in muscle fibers and widening of gaps; The scale in each image is 100 µm; (M) WB showed the protein expression of Slow MyHC, Fast MyHC, MuRF1, Atrogin1 and MSTN in muscles of air and CS-exposed mice; (N) the expression of Slow MyHC in muscles from mice. n = 5, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: CSE stimulation caused ferroptosis in C2C12 myotubes. (A) Cellular morphology of C2C12 myoblasts and C2C12 myotubes. The scale in each image is 100 µm; (B) the proportion of cell death was detected by flow cytometry with 7AAD staining in Ctrl and 3% CSE-stimulated myotubes; (C) RT-qPCR examined the expression of Gpx4, Slc7a11, Slc3a2, Tfr1, Ncoa4, Acls4 and Lpcat3 in C2C12 myotubes; (D) WB showed the protein expression of GPX4, MuRF1, Atrogin1, and MSTN in C2C12 myotubes; (E) the level of Fe2+ was detected by flow cytometry with the fluorescent probe; (F) the content of GSH in C2C12 myotubes; (G) lipid ROS was detected by flow cytometry with C11 BODIPY; (H) the level of LPO in C2C12 myotubes. (I) The proportion of cell death was detected by flow cytometry with 7AAD staining. UAMC-3203, a ferroptosis inhibitor. ***P < 0.001, ****P < 0.0001.