fig1

Figure 1. Establishment of NSCLC cell lines resistant to Osimertinib and Afatinib and studies on the key role of FAK in the acquired resistance to EGFR-TKIs in NSCLC. (A) H1975OR and PC9AR cells were established using a gradual stepwise selection protocol as illustrated in the figure [Created in BioRender. Xu, G. (2025)]; (B) H1975/H1975OR and PC9/PC9AR cells were treated with increasing concentrations of indicated drugs (Osimertinib or Afatinib) for 72 h. The growth rate was measured by the SRB assay. (IC₅₀ values were calculated by GraphPad Prism and represented as mean ± standard deviation, n = 3); (C) H1975 and H1975OR cells were cultured in 6-well plates and harvested for preparation of total RNA and subsequent gene expression analyses. The Volcano plot shows the differential expression of protein kinase genes between H1975 and H1975OR cells. Log2Foldchange > 1 or < -1 and Log10 (Padj) > 2 indicate significant differences; (D) KEGG pathway enrichment analysis of significantly DEGs and (E) PPI analysis for DEGs. The genes in EGFR-TKI resistance pathway are depicted in Red; (F) NSCLC cell lines were cultured in 6-well plates and harvested for protein and subsequent PAMchip analyses. The left figure panel shows the difference in phosphorylated protein kinases between H1975 and H1975OR cells. The right panel includes data from PC9 and PC9AR cells. A log2 fold change > 2 was selected as the cutoff value. Kinases with reduced activity are depicted in yellow, while kinases with enhanced activity are in blue; p-FAK is shown in Red. NSCLC: Non-small cell lung cancer; FAK: focal adhesion kinase; EGFR: epidermal growth factor receptor; TKIs: tyrosine kinase inhibitors; H1975OR: H1975 Osimertinib resistant; PC9AR: PC9 Afatinib resistant; SRB: sulforhodamine B; IC₅₀: half-maximal inhibitory concentration; KEGG: Kyoto Encyclopedia of Genes and Genomes; DEGs: differentially expressed genes; PPI: protein-protein interaction; p-FAK: phosphorylated focal adhesion kinase; Afa: Afatinib; Osi: Osimertinib; PI3K: phosphatidylinositol 3-kinase; MAPK: mitogen-activated protein kinase.