Figure 3. DFMO treatment decreases intracellular levels of spermidine. CHO cells were grown in full nutrient medium in the presence (+DFMO) or absence of 1 mM DFMO during 48 h and then were incubated for 2 h in control (Ctr) or starvation medium (Stv) or in control medium supplemented with 50 ng/μl rapamycin (Rap) alone (None) or in the presence of 100 nM wortmannin (+WM) or 1 mM DFMO (+DFMO). Intracellular pools of polyamines were determined by HPLC after benzoylation as indicated in Materials and Methods. Concentrations (ng /5 × 10 ⁶ cells) of spermidine (A) and spermine (B) are depicted for the indicated conditions. All values are the mean ± SD of two determinations. Significantly different from control: *p < 0.05, **p < 0.01. (C and D) CHO cells were grown in full nutrient media alone (None) or in the presence of 1 mM DFMO (+DFMO), 100 μM Spd (+Spd) or 1 mM DFMO plus 100 μM Spd (+DFMO +Spd) during 48 h and then were incubated for 2 h in control (Ctr) or starvation medium (Stv) followed by MDC labeling or immunodetection of endogenous LC3 protein. Percentage of cells with more than 10 MDC-labeled dots per cell (C) or 10 endogenous LC3 puncta per cell (D) was calculated for each condition. Data represent the mean ± SEM of at least three independent experiments (number of counted cells ≈100). Significantly different from control: *p < 0.05, **p < 0.01.