NIH-3T3 cells were transfected with a plasmid encoding either N-FLAG-MT1 or FLAG and treated with TGF-β1 (10 ng/ml) or vehicle. (A–E) qPCR analysis demonstrates minor but significant reduction of Postn expression in MT1-overexpressing cells but not other myofibroblast markers. Statistics were performed using 1-way ANOVA and Tukey’s post-hoc test (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Representative images of neonatal mouse CMs were treated with conditioned media from untransfected NIH-3T3 cells or cells expressing N-FLAG-MT1. Confocal immunofluorescent detection of FLAG localization reveals uptake of N-FLAG-MT1 by CMs (white arrows). Yellow lines on the xy plane indicate orthogonal planes outlined in white (yz and xz). Scale bars: 10 μm. Experiment was performed 4 independent times. (G) Neonatal mouse CMs were treated with indicated dose of H₂O₂, and cell number was assessed by CyQuant DNA-dye incorporation. H₂O₂ treated CM displayed reduced viability compared with untreated CMs at the indicated treatment time (n = 4 per data point). (H) Neonatal mouse CMs were treated with indicated concentration of H₂O₂ and recombinant MT1 or MT2A, followed by CyQuant DNA-dye incorporation to assess cell number. MT1 and MT2A were equally able to protect neonatal CMs from H₂O₂-induced cell death (n = 4 per data point). (I) Neonatal mouse CMs were cultured in the presence of 0, 125, or 250 μM H₂O₂ and 0, 500, 1000, 2000, or 5000 nM of recombinant rabbit MT2A. CyQuant DNA dye incorporation reveals a dose-dependent protection of CMs from cell death by MT2A (n = 12 per data point). (J) Treatment of adult CM with 5 μM MT1 or MT2A improved viability in vitro. Statistics in G–H were performed using 2-way ANOVA and Tukey’s post-hoc test (n = 4 per data point). *P < 0.05, ****P < 0.001, ****P < 0.0001 compared with 0 μM H₂O₂ within indicated MT2A treatment. ^##P < 0.01, ^####P < 0.0001 compared with 0 nM MT2A within indicated H₂O₂ treatment.