6-Thio-dG in combination with osimertinib synergistically induces apoptosis and TIFs in osimertinib-resistant EGFRm NSCLC cell lines. (A) The given cell lines were treated with varied concentrations of the tested agents either alone or in combinations for 3 days. Cell numbers were then measured by SRB assay and CIs were calculated and presented inside the graph. The data are means ± SDs of four replicate determinations. (B) The tested cell lines seeded in 12-well plates were treated with 50 nM osimertinib, 50 nM 6-Thio-dG (Thio), or their combination, which were repeated with fresh medium every 3 days. After 10 days, the cells were fixed, stained with crystal violet dye, imaged, and counted. Columns are means ± SDs of triplicate determinations. (C–G) The tested cell lines were exposed to 200 nM osimertinib, 250 nM 6-Thio-dG or their combination for 16 h (E), 24 h (C and F), or 48 h (D and G). The proteins of interest were detected with western blotting (E, C, and F) and apoptotic cells were detected with annexin V staining/flow cytometry (D and G). Each column represents mean ± SD of triplicate treatments. (H and I) Both PC-9/AR and HCC827/AR cells were treated with 200 nM osimertinib, 250 nM 6-Thio-dG, or their combination for 24 h followed by the TIF assay. TIFs were counted from 20 cells for each treatment and represented as means ± SEs. The inserted images show costaining of DAPI, TRF2 and γ-H2AX. Arrows indicate TIFs (colocalization of TRF2 and γ-H2AX). Statistical analysis was conducted with one-way ANOVA test (B, D, and I) or two-sided unpaired Student’s t test (G). Source data are available for this figure: SourceData F5.