Fig. 4

PFKFB3 inhibition triggers oxidative DNA damage in TKI-treated cells. A NSCLCs were exposed to the indicated treatments for 24 h. Immunocytochemical staining of oxidized guanine (8-OXO-G, green) and nuclear staining with DAPI (blue). Scale bar 100 μm. B Cells were analyzed using the CellProfiler pipeline; nuclei with 8-OXO-G punctate were normalized to the total nuclei amount in the field and presented in % (n = 45). C PC9 cells were transfected with PFKFB3 siRNAs (si#1, si#2) followed by treatment with either vehicle or erlotinib (PC9, 0.5 µM) for 24 h. Whole cell lysates were analyzed in immunoblotting with the indicated antibodies. β-actin was used as a loading control. Target ratios were quantified using densitometry and normalized to vehicle-treated samples. D PC9 cells were exposed to PFKFB3 inhibitors (PFK158, KAN757) or/and EGFR inhibitors (erlotinib, osimertinib) for 24 h. Whole cell lysates were analyzed by Western blotting with the indicated antibodies. β-actin was used as a loading control. Target/loading control ratios were quantified using densitometry and normalized to vehicle-treated samples. E MPG, NTHL1, and UNG2 protein levels in PC9 cells exposed to appropriate treatments were analyzed in immunoblotting in 3 independent experiments (n = 3). B, E Statistical analysis by ONE-WAY ANOVA with Tukey’s post hoc tests (p-values are shown as follows: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001). MPG - N-methylpurine DNA Glycosylase, NTHL1 - Nth Like DNA Glycosylase 1, UNG - Uracil-DNA glycosylase