Figure 6. Inhibiting PI3K, JNK or p38MAPK does not inhibit KRASG12V or BRAFV600E -inducible EGR1:EmGFP activation.
(A) HeLa cells containing the EGR1:EmGFP reporter construct and expressing Doxycycline-inducible KRASG12V (KR1) or BRAFV600E (BR5) were treated with 100 nM Trametinib (Tram), 200 nM ZSTK474 (ZSTK), 50 nM BIRB796 (BIRB), or 5 µM JNK inhibitor VIII (JNK). Cells were then treated with 100 ng/ml Doxycycline (Dox) or DMSO for 24 h, fixed and stained with DAPI before being imaged on a high content microscope. Results were analysed to determine the mean GFP intensity within the population. GFP intensity from parental cells was taken as background. Results represent the means ± SD from three independent experiments. (B) Cells were treated as above, and whole cell lysates fractionated by SDS–PAGE and blotted with the indicated antibodies. Results are representative of three independent experiments. (C and D) Quantitative western blot analysis of blots shown in figure (B). (C) p-MEK1/2 relative to MEK1/2 or (D) p-ERK1/2 relative to ERK1/2. Results are the mean normalised blot quantification ± S.D. from three independent experiments. P-values: nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 analysed by two-way ANOVA followed by Tukey's multiple comparison test.