Figure 1
Inhibitors of MAP4K4 confer protection from DOX in rat H9c2 cardiomyocytes. H9c2 cells were treated with the MAP4K4 inhibitors shown, or DMSO as the vehicle control, beginning 1 h prior to DOX. (A–C) Viability, measured by the CellTiter-Glo assay. (A,B) Dose–response for DOX cardiotoxicity at 24 h (A) and 48 h (B), in the absence or presence of 10 μM F1386-0,303, the second-generation MAP4K4 inhibitor11. Data are triplicates, plotted as the mean ± SEM, and are representative of 3 independent dose–response experiments. (C) Protection from 333 nM DOX at 48 h by F1386-0303 versus DMX-5804, a third-generation inhibition11. Data are from a single experiment, plotted as the mean ± SEM, and are representative of ≥ 20 independent dose–response experiments across multiple batches of each compound. (D–F) Activation of executioner caspases. (D,E) Dose–response for caspase-3/7 activity triggered by DOX, at 6 h (D) and 24 h (E), in the absence or presence of 10 μM F1386-0303. Data are triplicates, shown as the mean ± SEM. (F) Dose–response for protection from 1 μM DOX at 24 h by F1386-0303 versus DMX-5804. Data are the mean ± SEM for quadruplicate cultures in three independent experiments. (G–I) Preservation of mitochondrial membrane potential (TMRM fluorescence). DOX, 222 nM; DMX-5804, 10 μM; CsA, 2 μM. (G) Representative photomicrographs at 1 and 16 h. Upper rows, phase-contrast microscopy of the fields shown below, for reference. Lower rows, TMRM fluorescence. Bar, 100 μM. (H) Time-course of TMRM fluorescence, measured hourly. Data are quadruplicates, plotted as the mean ± SEM, and are representative of 3 independent time-course experiments. (I) Dose–response comparing protection from 222 nM DOX at 16 h by F1386-0303 (pEC50 5.9) versus DMX-5804 (pEC50 5.5). Data are plotted as the mean ± SEM from 3 independent dose–response experiments run in triplicate.
