Fig. 1.

Effect of mTOR overexpression and Evr treatment on the Warburg effect in GEM_sen and GEM_res cells. The transfection efficiency of mTOR overexpression (ov-mTOR) vectors was evaluated by measuring a the relative mRNA expression of mTOR and b the percentage of positive fluorescence signal (transfected cells) in GEM_sen and GEM_res cells. The transfection efficiency was determined to be approximately 37%. c Effect of ov-mTOR on the viability of GEM_sen and GEM_res cells, with or without Evr treatment. d Glucose uptake, e ATP production, and f lactic acid generation in GEM_sen and GEM_res cells with or without ov-mTOR transfection or/and Evr treatment. mTOR overexpression promoted glucose uptake, ATP production, and lactic acid generation in both cell types, but its effect appeared to be less pronounced in GEM_res cells than that in GEM_sen cells. g Western blot of proteins associated with the Warburg effect. Quantification of the protein expression of h GLUT1, i LDHB, j HK2, and k PKM2 in GEM_sen and GEM_res cells with or without ov-mTOR transfection or/and Evr treatment, normalized to that of GAPDH as an internal control. From h to k, the numbers in the arrows represent the difference (increase or decrease) in protein expression between ov-mTOR and ov-mTOR + Evr. In ov-mTOR-transfected GEM_res cells, the suppressive effect of Evr on the expression of GLUT1 and LDHB was weaker than that in ov-mTOR-transfected GEM_sen cells. Conversely, the suppressive effect of Evr on the expression of HK2 and PKM2 was stronger in ov-mTOR-transfected GEM_res cells than that in ov-mTOR-transfected GEM_sen cells. The data are expressed as the mean ± standard deviation of three replicates (n = 3). *P < 0.05; ^#P < 0.05 compared with the same treatment in GEM_sen cells. GEM: gemcitabine, Evr: everolimus; EV: empty vector (negative control); GEM_sen: GEM-sensitive pancreatic cancer cells; GEM_res: GEM-resistant pancreatic cancer cells; au: arbitrary units