(A) CCL-151, CCL-153, A549, and 16HBE14o- cells were exposed to tonic stretch (30% strain) for 4 hours, and then replicate analysis (validating the microarray analysis in Tables 1 and 2) of stretch-induced expression by real-time quantitative PCR (qPCR) for MT1G, MT1M, and MT1X transcript levels (n = 3) expressed relative to time-matched controls (n = 3) was performed. (B) CCL-151 cells were tonically stretched (30% strain) for the indicated periods of time before RNA harvest and gene expression analysis by qPCR. An independent 6-well BioFlex plate was used for each time point (n = 3 stretched and 3 control wells per time point). *P < 0.05 versus unstretched control for all 3 genes by Kruskal-Wallis test with Dunn’s multiple comparison’s test. (C) CCL-151 cells were exposed to tonic stretch with amplitudes of 10%, 20%, and 40% strain and assessed for transcript level changes after 4 hours. Each strain increment was measured from an independent 6-well BioFlex plate (n = 3 stretched and 3 control wells per increment of strain). *P < 0.05 versus unstretched control for MT1G by Kruskal-Wallis test with Dunn’s multiple comparisons test. (D) HMVEC-L cells and (E) CCL-151 fibroblasts were cyclically stretched 20% (sinusoidal waveform) at 0.33 Hz for 4 hours and probed for MT transcript levels by qPCR (n = 3 stretched and 3 time-matched controls for endothelial cells and n = 5 stretched and 5 time-matched controls for fibroblasts). *P < 0.05 versus control, Student’s 2-tailed t test. (F) CCL-151 cells subjected to cyclical stretch as in panel E or unstretched control cells (pooled from 3 separate wells) were harvested for protein and analyzed by Western blotting using MT and β-actin (loading control) antibodies. As a control to confirm specificity of the MT antibody, lungs harvested from WT and MT knockout (KO) mice subjected to ventilator-induced lung injury (VILI) for 4 hours with 24 ml/kg tidal volume, as described in more detail in Figure 2, were analyzed by Western blotting using the same antibodies.