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. 2016 Jan 9;7(7):7866–7884. doi: 10.18632/oncotarget.6872

Figure 10. ERK phosphorylation and levels of ERK/MDM2 complex in U87MG cells.

Figure 10

A. U87MG cells were treated with DMSO (control), 1 μΜ EB148 or 1 μΜ EB54 for the indicated times; in some experiments, after 2 h of treatment the medium was replaced by fresh medium not containing drugs for an additional 2 h. At the end of the treatment period the levels of ERK 1/2 phosphorylation were evaluated using ELISA kits, as described in the Methods section. The data are expressed as the percentage of phosphorylated ERK1/2 versus untreated cells (control), which were set at 100%. Data are the mean values ± SEM of three independent experiments performed in triplicate. Analysis of significant differences in mean ERK/MDM2 complex levels was performed using one-way ANOVA with a Bonferroni post-test: * p< 0.05, ** p< 0.01, *** p<0.001 vs. control. B. U87MG cells were treated for 30 min with DMSO (control) or the p53 inhibitor, pifithrin-β (1 μΜ); subsequently, cells were incubated with DMSO, EB148 or EB54 1 μM for 2 h. At the end of the treatment periods, the levels of ERK 1/2 phosphorylation were evaluated using ELISA kit, as in A. The significance of differences was performed using one-way ANOVA with a Bonferroni post-test: * p< 0.05 vs. control; #p<0,05 vs. cells not treated with the p53 inhibitor. C. U87MG cells were treated with DMSO (control), 1 μΜ EB148 or 1 μΜ EB54 for the indicated times; in some experiments, after 8 h of treatment, the medium was replaced by fresh medium not containing drugs for an additional 24 h. At the end of the treatment period, U87MG cells were collected and suspended in lysis buffer. Equal amounts of cell lysates were captured on wells pre-coated with MDM2 antibody. After extensive washing, the levels of the ERK/MDM2 complex were quantified using an antibody specific for ERKs, and subsequently an HRP-conjugated antibody and a TMB substrate kit. Data are expressed as percentage of control set to 100%, and represent the mean ± SEM of three independent experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: * p<0.05, ** p<0.01, *** p<0.001 vs. control.