Fig. 4
m5C reader YBX1 expression is required for intrinsic gefitinib resistance. a Sensitive and resistant cells were treated with gefitinib (1 µM) or osimertinib (1 µM) for 24 h and YBX1 protein level was examined by western blotting. b Sensitive and resistant cells were treated with gefitinib (1 µM) or osimertinib (1 µM) for 24 h and YBX1 mRNA expression was determined by qRT-PCR analysis. c, d Representative images of YBX1 IHC staining (c) and the quantitative H-scores (d) in pre-treatment and post-treatment biopsies of NSCLC patients with intrinsic resistance to gefitinib. Image magnification: 200 × (upper panel) and 400 × (lower panel). e, f H1650 and H1975 cells transfected with siRNA targeting YBX1 (siYBX1) or non-targeting control (siCtrl) were exposed to gefitinib (1 µM) for 72 h and cell viability (e) or cell apoptosis (f) was respectively detected by CCK-8 assay or Annexin V-FITC/PI staining. g H1975 cells transfected with siYBX1 was detected for EGFR protein expression by western blotting analysis. h, i H1650 cells pre-treated with shYBX1 were stably transfected with wild type YBX1 (YBX1-WT) or the binding deficient mutant (YBX1-Mut, W65A) and cell proliferation was detected by CCK-8 (h) or colony formation assay (i). j, k Tumor growth (j) and tumor weights (k) of H1650-shCtrl and H1650-shYBX1 cells-derived xenografts subcutaneously implanted in BALB/c nude mice (n ≥ 5). l Representative images of ki67 IHC staining and the quantitative H-scores of tumors obtained from H1650-shCtrl and H1650-shYBX1 xenografts. Image magnification: 200 × (upper panel) and 400 × (lower panel). Data are represented as means ± SD. For a, b, e-f, n = 3 biological independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.