FIG. 3.

ASO-induced skipping of exons 16, 18, and 21 results in reduced in vitro cell viability and EGFR signaling inhibition in H292, PC9, and PC9-GR cells. Cell viability was assessed in (a) H292, (b) PC9, and (c) PC9-GR cell lines measured using Cell Titer Glo Assay (Promega) and revealed that ASO targeting of exon 18 resulted in the largest reduction in cell viability across all three cell lines, whereas erlotinib only reduced viability in PC9 cells. Western blot analysis of (d) PC9 cells and (e) PC9-GR cells revealed that ASOs directed to the tyrosine kinase domain (exons 18 and 21) resulted in more profound suppression of EGFR signaling than targeting the transmembrane domain (exon 16). Blots show results for phosphorylation of EGFR on tyrosine (Y) 1,068, Akt at serine (S) 473, Erk targets at threonine (T) 202/tyrosine (Y) 204, and S6 at serine (S) 235/236. Cells were treated with 5 μM concentrations of the indicated ASOs and 100 nM erlotinib (ER). Neg1 and Neg2 are custom negative controls, and STD refers to the company-provided standard negative control. All the treatments lasted 48 h. Asterisks indicate two-tailed t-test significance using thresholds of *P < 0.05, **P < 0.01, and ***P < 0.001.