Fig. 7.

GFPT2 is a target of nutlin-3a-induced cell death. GFPT2 was inhibited by exogenously introduced siGFPT2 for indicated times in KRAS MT/p53 WT NSCLC cells (A-F). A, B Expression of GFPT2, O-GlcNAc (A), and the KRAS-PI3K/Akt-mTOR pathway (B) were detected by western blotting. C KRAS-GTP form was detected through the KRAS activation assay. D Cell viability was measured by MTT assay. E Apoptotic cell death number (top), and total cell death number (bottom) were analyzed via flow cytometry using annexin V/PI staining. F Expression of caspase-9, caspase-3, and PARP detected by western blotting. Etoposide (ETO) treatment (100 μM) for 24 h was used as a positive control for inducing caspase cleavage. GFPT2 was re-expressed by transfection of GFPT2-HA for 24 h into GFPT2 knockout A549 cells (G-L). G KRAS-GTP form and O-GlcNAc were verified by KRAS activation assay and western blotting. H, I Cell viability was detected by cell counting (H), and MTT assays (I). J, K Cytoplasmic vacuoles were examined under the light microscope (J), and confocal microscope after incorporation of dextran (green) (K). L Autophagic flux was detected after transfection of the mRFP-GFP-LC3 tandem vector (n ≥ 10). *p < 0.05, **p < 0.01, ***p < 0.001 compared with control