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. Author manuscript; available in PMC: 2020 May 1.

Published in final edited form as: J Thorac Oncol. 2019 Mar 1;14(5):802–815. doi: 10.1016/j.jtho.2018.12.038

FIGURE 2. — A. CRISPR/Cas9 genome engineering protocol scheme. B. Expression of AGK/BRAF. C. Sanger sequencing of PCR product confirming fusion in cell line of AGK exon 2 with exon 8 of BRAF. D. H1975 and PC9 cells expressing AGK/BRAF were treated with 0.5 osimertinib for 1 h, whole-cell extracts prepared and then lysates were subjected to immunoblotting. Results represent 3 independent experiments. E. The indicated cell lines were plated at a density 7,500/well in 96-well plates, treated with osimertinib for 96 h and the IC₅₀ values for growth inhibition are shown in the Table in panel F. G-H. Cells were plated at a density of 150,000 cells/well in 6-well plates then infected 24 h later with lentiviruses harboring the indicated shRNAs and then selected with puromycin 48 h later. BRAF mRNA levels was determined by qRT-PCR (G) or cells were replated in osimertinib and growth assessed 96 later (H). IC₅₀ values for growth inhibition were determined by nonlinear regression analysis using Graphpad Prism software. Results represent the mean ± SD of 2 experiments.

FIGURE 2. — A. CRISPR/Cas9 genome engineering protocol scheme. B. Expression of AGK/BRAF. C. Sanger sequencing of PCR product confirming fusion in cell line of AGK exon 2 with exon 8 of BRAF. D. H1975 and PC9 cells expressing AGK/BRAF were treated with 0.5 osimertinib for 1 h, whole-cell extracts prepared and then lysates were subjected to immunoblotting. Results represent 3 independent experiments. E. The indicated cell lines were plated at a density 7,500/well in 96-well plates, treated with osimertinib for 96 h and the IC₅₀ values for growth inhibition are shown in the Table in panel F. G-H. Cells were plated at a density of 150,000 cells/well in 6-well plates then infected 24 h later with lentiviruses harboring the indicated shRNAs and then selected with puromycin 48 h later. BRAF mRNA levels was determined by qRT-PCR (G) or cells were replated in osimertinib and growth assessed 96 later (H). IC₅₀ values for growth inhibition were determined by nonlinear regression analysis using Graphpad Prism software. Results represent the mean ± SD of 2 experiments.