Way (as determined by the Sanger Cell Line Project and also the Cancer Cell Line Encyclopedia [Barretina et al., 2012]). Experiments had been done in biological duplicate using the typical values presented EM. (B) Crystal Violet stain of cells plated within the indicated doses of BCI or manage (0 = 0.1 DMSO) for 72 hr. Sensitive cells with a KRAS mutation (H358 cells; denoted with red underlining) show a much more pronounced decrease in cell number than do cells with out oncogenic HDAC6 Inhibitors medchemexpress mutations in genes encoding components of your EGFR-KRAS-ERK pathway (H1648 cells; black underlining). Experiments were accomplished in biological duplicate using a representative image shown. (C) BCI increases P-ERK levels especially in BCIsensitive cell lines. Sensitive lines (H358, PC9, H1975 and A549; red underlining) and insensitive lines (HCC95 and H1648; black underlining) were treated with the indicated doses of BCI or vehicle control (0.1 DMSO) for 30 min, along with the levels of ERK (p44/p42) and P-ERK (P-p44/42 T202/Y204) Figure four continued on subsequent pageUnni et al. eLife 2018;7:e33718. DOI: https://doi.org/10.7554/eLife.10 ofResearch article Figure 4 continuedCancer Biologyassessed by Western blot. P-ERK appeared within the sensitive cells at low doses of BCI, but P-ERK levels did not increase within the insensitive cells in the tested doses of BCI. (D) Dosimetry plots from the experiment shown in panel. (C) (E ) Cell lines sensitive to BCI are also dependent on P-ERK for survival. BCI-sensitive cells with oncogenic mutations in EGFR or KRAS (PC9 and H358, respectively; red underlining) and BCI-insensitive cells (H1648 and HCC95; black underlining) were treated with the indicated doses with the MEK inhibitor trametinib for 72 hr; viable cells were measured with Alamar blue and in comparison with cells getting the vehicle handle (0 = 0.1 DMSO). (E) Treatment with trametinib decreased P-ERK levels as determined by western blot. (F) The reduction in P-ERK corresponded to a higher decrease in viable cells in BCI-sensitive lines (red coloring), in comparison with BCIinsensitive cell lines (black coloring). DOI: https://doi.org/10.7554/eLife.33718.008 The following figure supplement is available for figure 4: Figure supplement 1. (A ) Knockdown of DUSP6, but not DUSP1, decreases viability of LUAD cells. DOI: https://doi.org/10.7554/eLife.33718.uM), contains an activating mutation in MEK (Q56P); and three) the comparatively insensitive lines (IC50s ! five uM) lack known mutations affecting the EGFR-KRAS-ERK signaling pathway. The insensitive cell lines didn’t demonstrate the marked ( 90 ) reduction in numbers of viable cells observed with all the sensitive cell lines and only sensitive cell lines showed induction of cleaved PARP soon after BCI remedy (Figure 4–figure supplement 1C). Together, these data recommend that pharmacological inhibition of DUSP6 particularly kills cells with EGFR or KRAS-mutations.P-ERK levels enhance in LUAD cells following inhibition of DUSP6 by BCI, and P-ERK is required for BCI- mediated toxicityBased on findings inside the preceding section, we predicted that BCI-mediated inhibition of DUSP6 would enhance P-ERK to toxic levels, comparable for the effects of co-expressing mutant KRAS and EGFR. To test this proposal, we measured total ERK and P-ERK immediately after BCI remedy in sensitive and insensitive cell lines. A subset with the most sensitive cell lines, H358 (KRAS mutant) and PC9 and H1975 (EGFR mutants), demonstrated a sizable, Methyl 3-phenylpropanoate Purity & Documentation dose-dependent boost in P-ERK in response to BCI therapy, with appreciable incr.