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. 2016 Feb 15;6:20831. doi: 10.1038/srep20831

Figure 1. Cobinamide Rescue of Cellular Sulfide Toxicity.

Figure 1

(a) Oxygen consumption was measured in hiPSC-derived neurons; 1 mM NaSH and/or 100 μM cobinamide (Cbi) were added as indicated. Control cells (circles, solid grey line); cobinamide-treated cells (squares, dashed grey line); NaSH-treated cells (triangles, solid black line); NaSH-treated cells subsequently treated with cobinamide (triangles, dashed black line). (b) Permeabilized human fibroblasts were incubated with TMPD, a complex IV substrate; 2 μM rotenone, 4 μM antimycin A, 0.5 mM NaSH or KCN, and/or 100 μM cobinamide were added as indicated. Oxygen consumption was measured as in a, but reflects largely complex IV activity. Symbols are as in (a) except KCN-treated cells (triangles, solid grey line) and KCN-treated cells that received cobinamide (triangles, dashed grey line) are shown. (c) Extracellular acidification was measured in human fibroblasts, with 1 mM NaSH and 100 μM cobinamide added as indicated. Symbols are as in (a). (d) Cobinamide (1 mM) was added to water or DMEM culture medium with or without an equimolar amount of NaSH. The samples were diluted 20-fold and their UV-visible spectra recorded. (e–g) hiPSC-derived neurons (e) or human fibroblasts (f,g) received 1 mM NaSH or 1 mM KCN for 2 h, with some cells receiving 400 μM cobinamide or 200 μM 4-hydroxy-TEMPO (Tem) during the second hour. The cells were washed and incubated in non-drug-containing medium. (e) At 20 h, cells were stained with DAPI (nuclei, blue) and β3-tubulin (green); TUNEL-positive, apoptotic cells were stained red. Numbers below the photographs are the percent of apoptotic cells (mean ± SEM); scale bars are 50 μm. (f) At 24 h, cells were counted, and cell doublings are shown as a percent of control cells. (g) At 24 h, DNA synthesis was measured. Con, control, Veh, vehicle. The data are the means ± SEM of three independent experiments performed in triplicate. In (a–c) the difference between cobinamide-treated and NaSH or KCN only was significant within 8 min post cobinamide addition (P < 0.05). In e, ***P < 0.001 for comparison to control or NaSH-treated cells, and in f and g, *P < 0.05, **P < 0.01, ***P < 0.001 for indicated comparisons.