Laminin

7-AAD and Annexin-V-FITC were from Cayman Chemical (Ann Arbor, MI, USA). Mouse IFN-γ was from eBiosciences (San Diego, CA, USA). LPS and oligomycin were from Sigma Aldrich (St. Louis, MO, USA). JC-1, MitoSOX and CellROX Green were from Molecular Probes, Life Technologies Corporations (Grand Island, NY, USA). Rabbit polyclonal anti-phospho-p70S6K-T389, laminin, HIF-1alpha, phospho-Akt-S473 and p70S6K were from Cell Signaling Technologies (Danvers, MA, USA). Goat polyclonal anti-Akt and rabbit polyclonal anti-Actin were from Santacruz Biotechnologies (Dallas, TX, USA). Goat anti-Rabbit IgG-IRDye 800CW and Donkey anti-Goat IgG-IRDye 800CW were from LI-COR BioSciences (Lincoln, NE, USA).

After washing twice with cold PBS, TMCs were lysed in RIPA buffer (cat. no. E-BC-R327; Elabscience, Wuhan, China) to extract total protein. The protein samples (30 μg) were denatured and separated by 10% SDS/PAGE. Protein was then moved onto PVDF membranes and blocked with 5% skimmed milk containing PBS to block nonspecific binding. Subsequently, the membranes were cultured overnight at 4°C with primary detection antibodies, including cleaved caspase-3 (ab32042; Abcam, Cambridge, USA), cleaved caspase-9 (# 20750S, Cell Signaling Technology, MA, USA), collagen I (ab138492), fibronectin (ab268020), laminin (ab108536), SMAD2 (ab40855), TGF-β (ab215715), and β-actin (# 4970S; Cell Signaling Technology). Appropriate secondary antibodies were then incubated with the blots for 1 h at room temperature. After washing, the signals were monitored with ECL Advance reagents (GE Healthcare, Braunschweig, Germany) and analyzed with Image Lab v6.0 software.