Fig. 5: NNMT promotes glycolysis and EGFR-TKIs resistance via SIRT1-mediated c-myc deacetylation.

A, B PC9 and HCC827 cells were stably transfected with NNMT-overexpressing vector (A), PC9/GR or HCC827/GR cells were transfected with NNMT shRNA or control shRNA (B), the expression of STAT1 and β-actin were measured by Western blot. C, D PC9 and HCC827 cells were stably transfected with NNMT-overexpressing vector (C), PC9/GR or HCC827/GR cells were transfected with NNMT shRNA or control shRNA (D), the SIRT1 activity levels were determined using a SIRT1 deacetylase fluorometric reagent kit. E Total protein extracts of PC9/GR cells that stably express NNMT or a control vector were subjected to IP using c-myc antibodies, followed by IB with Acetylated-Lysine Antibody and c-myc antibody. Reciprocal IP was done using Acetylated-Lysine Antibody, followed by IB with the c-myc antibody. F PC9/GR cells were co-transfected with NNMT shRNA or control shRNA and pCMV-SIRT1, the protein extracts were subjected to IP using c-myc antibodies, followed by IB with Acetylated-Lysine Antibody and c-myc antibody. Reciprocal IP was done using Acetylated-Lysine Antibody, followed by IB with the c-myc antibody. G, H Glucose uptake and Lactate secretion levels in PC9/GR cells co-transfected with NNMT shRNA or control shRNA and pCMV-SIRT1. I PC9/GR and HCC827/GR cells co-transfected with NNMT shRNA or control shRNA and pCMV-SIRT1 were treated with gefitinib at the indicated concentration for 72 h, cell viability was evaluated by MTS assay. Each point represents the mean ± SD. Data show a representative of three independent experiments. (*p < 0.05, **p < 0.01).