Figure 1.

U0126 protects PC12 cells against H₂O₂-induced cell death. (a) Chemical structures of U0126, its inactive analogue U0124, and other MEK inhibitors used in the study: trametinib, CI-1040, PD318088, and pimasertib. (b) Representative images of H₂O₂-induced dead cells stained by propidium iodide. These are accompanied by bright field images that show all cells. Scale bar = 100 μm. (c) U0126 treatment shows reduced cell death compared with DMSO (p < 0.002) and significantly lower death rate compared with other MEK inhibitors. U0124 shows similar, albeit to a lesser extent, protective effect. Control anti-oxidants, trolox and ascorbic acid, demonstrate significant protective effect toward oxidative stress. For each condition, the cell death percentage is computed from 75 individual images taken from 3 independent sets of experiments and each image comprises 150–250 cells (∼100 000 total number of cells). SEM error bars are depicted in the graph. (d) U0126 shows protective effect against oxidative stress induced by 0.01 units of glucose oxidase in glucose-supplemented medium for 12 h, while MEK inhibitors demonstrate similar high cell death rates as to the DMSO control. Trolox and ascorbic acid demonstrate significant protective effect toward oxidative stress. (e) Western blot of ERK phosphorylation demonstrates that the MEK inhibitors U0126, trametinib and pimasertib are effective in blocking ERK phosphorylation under complete media (left panel), while U0124 results in slight ERK inhibition compared to DMSO control. Serum starvation results in lack of ERK phosphorylation in all conditions. (f) The cell-protective effect of U0126 is concentration dependent and shows an EC₅₀ of about 100 nM.