Fig. 1: RAF inhibitors drive the rapid inhibition of DNA replication independent of paradoxical ERK1/2 activation.
From: RAF inhibitors activate the integrated stress response by direct activation of GCN2
a NCI-H358 cells treated with the indicated concentrations of Dabrafenib for 24 hours with a 10 μM EdU pulse for the final hour. Cells were then fixed and permeabilized for EdU detection (red line), immunofluorescence with a p-ERK1/2-specific antibody (blue line) and co-stained with DAPI. Mean signal per cell was determined by high-content image analysis of 2000-15000 cells per condition and normalised to DMSO control. Normalised mean values ± SD are shown, n = 3 biological replicate experiments (b, c) NCI-H358 cells were treated with 1 (red triangle), 3 (green diamond), 5 (brown diamond) or 10 nM (dark blue triangle) Trametinib for 1 hour prior to addition of the indicated concentration of Dabrafenib for 24 hours. Some cells received increasing concentrations of Trametinib alone for 24 hours (black triangle). p-ERK1/2 (b) and EdU (c) incorporation were quantified by HCM analysis as above. Results shown are mean ± SD of n = 2 (pERK1/2) or 4 (EdU) biological replicate experiments. d–f NCI-H358 cells were treated with 1 µM Dabrafenib (blue square), 1 µM PLX-7284 (red triangle), 10 µM Vemurafenib (green triangle), 100 nM Trametinib (black triangle) or 10 µM Etoposide (purple triangle) for the indicated times and analysed for p-ERK1/2 (d), percentage of EdU-postive cells (e) or intensity of EdU staining (f) as above. Results mean ± SD of n = 3 biological replicate experiments.
