Figure 3

Effects of acute Selumetinib treatment on ERK1/2 activity in colorectal tumour biopsies. (a–e) Twenty three tumour biopsies were treated for 1 h ex vivo with 0, 0.1 or 3 µM Selumetinib, as outlined in Fig. 2a. Western blot analysis with antibodies to phosphorylated ERK1/2 (pERK1/2), native ERK1/2 and actin determined effects of the agent on relative ERK activity. A time 0 control was also analysed to gain information on baseline levels of active kinase. Quantification of western blots was carried out using ImageJ as described in materials and methods. (a) Graph showing mean (+/− s.e.m) relative p-ERK1/2 intensity at each dose N = 21. (b) Tumours were stratified based on fold decrease in relative pERK1/2 at 0.1 µM Selumetinib (compared to 0 µM). Stratified tumours were renumbered 1 to 23. ND = non-detectable at all doses. Those tumours mutant for KRAS or BRAF are highlighted in bold (see Fig. 5) (c) Example immunoblots. T3 and T14 have a > 2 fold decease in relative p-ERK1/2 levels following 1 h treatment with 0.1uM Selumetinib. T20 and T21 show minimal effect at this dose. Cropped gels are shown. Full length versions are in Supplemental Fig. S4c (d) Relative p-ERK1/2 levels at time 0 (basal) is shown for each tumour. (e) Effects of culture on relative p-ERK1/2 activity was determined by comparing intensity at T0 (basal) to 0uM (1 h culture) Mean of 23 samples (+/− s.e.m.) is shown.