Fig. 1

m⁵C hypermethylation and stable NSUN2 expression are linked to intrinsic gefitinib resistance in NSCLCs. a NSCLC cells were treated with gefitinib or osimertinib at gradient concentrations for 72 h and the IC₅₀ values were measured using CCK-8 assay. b Sensitive and resistant cells were treated with gefitinib (1 µM) or osimertinib (1 µM) for 24 h and the levels of 5-methylcytosine (m⁵C) in purified mRNA were measured by ELISA. c Sensitive and resistant cells were treated with gefitinib (1 µM) for 24 h and heatmap for gene expression of RNA m⁵C methyltransferases and demethylase from microarray analysis. Asterisk indicates control vs gefitinib group statistically significant difference. d Sensitive and resistant cells were treated with gefitinib (1 µM) or osimertinib (1 µM) for 24 h and mRNA expression of NSUN2 was detected by qRT-PCR. e Sensitive and resistant cells were treated with gefitinib (1 µM) or osimertinib (1 µM) for 24 h and the whole cell lysates were analyzed by western blotting with indicated antibodies. f, g Representative images of NSUN2 and p-EGFR IHC staining (f) and the quantitative H-scores (g) of pre-treatment and post-treatment biopsies of NSCLC patients with intrinsic resistance to gefitinib. Image magnification: 200 × (upper panel) and 400 × (lower panel). h Gene expression analysis of correlation between NSUN2 and drug resistance markers or tumor suppressors in TCGA-LUAD dataset. Data are means ± SD of three independent experiments. ns, not significant, *p < 0.05, **p < 0.01. Ctrl, control; Gef, Gefitinib; Osi, Osimertinib.