Figure 6: Co-targeting MEK1/2 and ERK5 pathways inhibits tumour cell growth.
(a) HCT116 cells were plated at 5 × 103 cells per well and treated with PD0325901 (0.63, 1.25, 2.5, 5.0, 10, 20, 40, 80, 160, 320, 640 or 1280, nM) with or without XMD8-92 (0, 1 or 10 μM). Cell viability was assessed by XTT assay after 48 h of treatment. (b) HCT116 cells were plated at 2 × 103 cells per well and cultured in the presence of DMSO, PD0325901 (100 nM), XMD8-92 (10 μM) or PD0325901 (100 nM)+XMD8-92 (10 μM). Compounds were added on day 0 and day 2. XTT assay was performed on five consecutive days. Results are normalized to cell densities on day 1 (n=8 per condition). (c) Small intestinal organoids were generated from WT or Apc−/− mice (first two lanes). Some Apc−/− organoids were transduced with KRASG12V lentivirus (third lane). Organoids were then analysed for the indicated signal transducers by western blotting. (d,e) Intestinal organoids generated from Apc−/− mice were treated with DMSO, PD0325901 (20 nM), XMD8-92 (10 μM) or PD0325901 (20 nM)+XMD8-92 (10 μM) for 5 days. Organoids were visualized by live-cell brightfield microscopy at the end of treatment (d), and analysed by Q-PCR (n=3 per condition) for expression of Mki67, c-Myc, Fra1 and c-Fos transcripts, normalized to Gapdh (e). (f,g) Intestinal organoids generated from Apc−/− mice were transduced with KRASG12V lentivirus, and treated with MAPK inhibitors as in d. The resultant Apc−/−;KRASG12V organoids were analysed by live-cell brightfield microscopy (f) and Q-PCR (n=3 per condition) for expression of Mki67, c-Myc, Fra1 and c-Fos transcripts, normalized to Gapdh (g). All data are mean±s.e.m. *P<0.05, **P<0.001 versus DMSO, ¶P<0.01 for PD0325901 versus PD+XMD (b), *P<0.05, **P<0.001 versus DMSO or as indicated, by analysis of variance (e,g). Scale bars, 100 μm (d and f).
