Figure 2.

Lung fibroblasts of IPF patients exhibit mitochondrial dysfunction. (A) Superoxide production as detected using the MitoSOX fluorophore (Red) in IPF and Ctrl‐LFs. Nuclei are counter‐stained using DAPI (blue). (B) Levels of mitochondrial superoxide, cellular ROS, mitochondrial DNA and stress (n = 4‐7). Mitochondrial stress was evaluated using the NAO fluorophore and microplate fluorometry. (C) Mitochondrial staining using MitoTracker Green in three separate cultures of IPF‐ and Ctrl‐LFs. (D) Expression of mitochondrial‐associated genes (n = 4‐7). mRNA data was normalized to GAPDH mRNA with Ctrl‐LF levels (2^{−Δ CT} x 10³) for PGC‐1α, PGC‐1β, UQCRC2 and NDUFB8 being 12 ± 7, 368 ± 40, 24 ± 2 and 40 ± 4, respectively. (E) mTORC1 activity was measured by increases in the phosphorylation of the mTORC1 substrate, p70S6k. Left top Immunoblot detection of pp70S6K in cell lysates obtained from LFs of separate IPF and Ctrl donors. Left bottom Fluorescent detection of total protein in gels before immunoblotting to verify protein loading. Right Quantitative data. *P < 0.05, **P < 0.01 compared to Ctrl‐LFs