Fig. 6

Reduced de novo nucleotide synthesis and oxidative stress contribute to cell death in NSCLCs under EGFRi therapy. A Scheme of [U-13C]-glucose utilization in PPP pathway. PC9 cells were exposed to appropriate treatments for 36 hours (including 24h exposure to tracer - [U-13C]-glucose.) B Fractional enrichment of M + 5 labeled nucleotides and nucleosides presented. Values represent biological replicates from 2 independent experiments (n=5-12). Data analyzed by TWO-WAY ANOVA with Tukey’s post hoc tests (p-values are shown as follows: *, <0.05; ***, <0.001; and ****, <0.0001.) C PC9 cells were exposed to appropriate treatments for 24h, and whole cell lysates were analyzed in immunoblotting. Glucose-6-phosphate dehydrogenase (G6PD) expression was normalized to β-actin (loading control). Target/loading control ratios were quantified using densitometry and normalized to vehicle-treated samples. D PC9 and HCC827 cells were exposed to assigned treatments for 24h with nucleotide pool (NTPs) replenishment in the last 6 hours. Cell viability was evaluated by trypan blue exclusion and normalized to vehicle-treated cells (DMSO). E PC9, HCC827, and H1975 cells were exposed to assigned treatments for 24h in the presence of antioxidant N-acetylcysteine (NAC, 5mM, 24h). Mean ± S.E. of 3 independent experiments presented (n=12). Statistical analysis by TWO-WAY ANOVA with Tukey’s and Śidak’s post hoc tests (p-values presented as follows: ##, <0.01; ###, <0.001, and ####, <0.0001, when compared to appropriate DMSO control. When comparing NTPs or NAC effect within the same treatment regimen, p-values are shown as follows: *, <0.05; **, <0.01; and ****, <0.0001). 6PG – 6-phosphogluconolactone