Figure 4. DFMO affects autophagosome formation. CHO cells were grown in full nutrient medium in the presence or absence of 1 mM DFMO (+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); 100 nM bafilomycin A₁ (+BAFA1) or 1 mM DFMO (+DFMO) as indicated in Materials and Methods. Afterwards, cells were lysed with sample buffer and the samples were subjected to western blot analysis to detect endogenous LC3 and TUBULIN proteins as indicated in Materials and Methods. (A) Representative immunoblots are depicted. (B) Densitometry was performed using NIH ImageJ. The LC3-II/TUBULIN normalized to control and (C) the LC3-II/LC3-I ratios were calculated. Data represent the mean ± SEM of four independent experiments. Significantly different from control: ***p < 0.001, *p < 0.05. (D) To evaluate autophagic flux, similar experiments were performed in the presence or absence of 100 nM BAFA1, which blocks lysosomal degradation. (E) LC3-II/TUBULIN ratio was calculated. Data are representative of three independent experiments. Significantly different from control: **p < 0.01, ns: non-significant.