FIGURE 7.

2-DG treatment increases the phosphorylation ratio of N-Myc in Thr-58. A, Western blot results for total and phosphorylated AKT, GSK3β, and N-Myc. GAPDH (not shown) was used for normalization. Samples were obtained and assayed in triplicate. Densitometry analyses were made with Image Lab^TM software. Phosphorylation ratios were calculated as the ratios phospho_treatment/total_treatment and phospho_control/total_control. Resulting data were analyzed with a one-sample t test (hypothetical mean = 1, 95% confidence interval). B, overexpression of n-myc in SK-N-SH cells. Cells were transfected with empty or n-myc-containing pcMV6XL4 vector and incubated for 24 h. Then medium was changed, and 3 mm 2-DG was added for a further 24 h. Western blot results for total and phosphorylated N-Myc are shown. GAPDH (not shown) was used for normalization. Samples were obtained and assayed in triplicate. Densitometry analyses were made with Image Lab^TM software. Phosphorylation ratios were analyzed with a one-sample t test (hypothetical mean = 1, 95% confidence interval). C, overexpression of n-myc increases the sensitivity of PA metabolism to 2-DG in SK-N-SH cells. Experiments were performed in triplicate. PAs were extracted from frozen cell pellets, derivatized with dansyl-chloride, and analyzed by HPLC. Statistic analyses were with Student's unpaired t test: 95% confidence interval; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Error bars, S.D.