(e) U0126 results in slightly lower cell death than DMSO upon treatment with 3.3 M cisplatin for 24 h. as a MEK inhibitor and for interpreting some previous results. < 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 150C250 cells (100?000 total number PF-06855800 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. We first use H2O2 as the oxidative stress inducer. We found that both the H2O2 concentration and the cell-plating PF-06855800 density drastically affected H2O2-induced cell death (Supporting Information Figure 1a). We screened series of H2O2 concentrations and cell densities and chose a condition (10 M H2O2 and 100?000 cells/well for 1 h) under which the control cells showed significant (40%) but not complete cell death. The low cell density was used to minimize cell-to-cell contacts and dead cells were determined by propidium iodide (PI) staining. Twelve-well plates were used so that different drug experiments were carried out at the same conditions using the same batch of cells. Automated fluorescence imaging (an automated scanning of 25 images per culture well) and automated image analysis (ImageJ) were carried out to avoid any potential bias in quantifying the data. Figure ?Figure1b,c1b,c shows that most of the MEK inhibitors induce similar or PF-06855800 higher cell death rates than the DMSO control, while U0126 treated cultures show drastically less PF-06855800 cell death. While this is in agreement with some previous studies that show U0126s protective effect,18,21 previous studies have attributed the protective effect of U0126 to the inhibition of the Raf/MEK/ERK pathway. On the other hand, our results clearly show that the protective effect of U0126 is not due to its function as a MEK inhibitor, as all four other MEK inhibitors fail to show any protective effect. This is supported by the observation that PF-06855800 its inactive analogue U0124 also protects against H2O2-induced cell death, Rabbit Polyclonal to 5-HT-6 albeit to a lesser degree. Antioxidants trolox and ascorbic acid are able to drastically suppress the cell death rates, highlighting that the cause of death for the PC-12 cells is due to the oxidative stress from H2O2. Further control experiments involving serum-starved PC12 cells without H2O2 treatment and nonstarved PC12 cells treated with 10 M H2O2 result in very little cell death (Supporting Information Figure 1b). We note that nonstarved PC12 cells show cell death upon treatment with H2O2 at a much higher concentration of 1 1 mM. We then use the glucose/glucose oxidase system to generate slow release of hydrogen peroxide in cells and in vitro instead of bolus addition.28 Upon treatment with glucose/glucose oxidase (0.01 units) for 12 h, the control cells show cell death at 71.2%. In contrast, the U0126 treated cells show significantly reduced cell death at 24.1%. Again, the MEK inhibitors CI-1040, pimasertib, and trametinib fail to show any protective effect, while the antioxidants trolox and ascorbic acid show drastic cell protection (Figure ?(Figure1d).1d). U0124 is also cell protective but not as effective as U0126. From both the H2O2 and glucose/glucose oxidase experiments, U0126 shows clear and consistent protective effects against H2O2-induced cell death, while other MEK inhibitors do not, indicating that the protective effect of U0126 is independent of its ability to inhibit the Raf/MEK/ERK pathway. We confirm that U0126 and other MEK inhibitors prevent the activation of Raf/MEK/ER signaling pathway by antiphospho-ERK Western blot. As shown in Figure ?Figure1e1e (left panel), PC12 cells cultured in serum-containing medium show.