Figure 4.

Reducing matrix stiffness sensitizes lung myofibroblasts to apoptosis by promoting MDM4–p53-dependent Fas expression. (A) Human IPF lung myofibroblasts were cultured on 1–20-kPa PA gels. Levels of Fas mRNA were determined by qPCR. (B) Levels of Fas protein were determined by immunoblot. (C–E) IPF lung myofibroblasts were cultured on 1- or 20-kPa PA gels in the presence of 50 nM MDM4 siRNA or the control siRNA (C), MDM4-expressing lentiviral vector or the control lentiviral vector (D), and 25 µM C646 or the vehicle (Vehi) control (E). Levels of Fas protein were determined by immunoblot. (F) IPF lung myofibroblasts were cultured on 1–20-kPa PA gels. Cell apoptosis was evaluated by TUNEL staining and IF/differential interference contrast (DIC) microscopy. Nuclei were stained by DAPI. A representative image shows cell apoptosis (arrowheads) under 20-kPa gel conditions. (G) IPF lung myofibroblasts were cultured on 1–20-kPa PA gels in the presence or absence of CH11 (100–500 ng/ml), MDM4-expressing lentiviruses or the control lentiviruses, C646, and MDM4 siRNA (siR) or the control siRNA. Cell apoptosis was evaluated by TUNEL staining and confocal IF microscopy. Nuclei were stained by DAPI. Representative images showed cell apoptosis (arrowheads) under 1-kPa gel conditions in the presence of 250 ng/ml CH11. (H) qPCR analysis for transcription of BAX, BID, NOXA, and PUMA in IPF lung myofibroblasts cultured on 1- and 20-kPa PA gels. GAPDH was used as internal reference control in qPCR analysis and as loading control in immunoblot analysis. Results are the means ± SD of three to five separate experiments. Apoptosis was evaluated in at least 200 lung myofibroblasts per experiment. Scale bar = 250 µm (F) and 50 µm (G). *, P < 0.05; **, P < 0.01 (ANOVA). k, kilodalton.