Research Paper Volume 13, Issue 17 pp 21483—21496

Figure 2. DMC increased the marker proteins of autophagy and autophagosomes in NPC cells. (A) Representative images of LC3-labelled red fluorescence puncta after 20 μM DMC for 0, 24, 48 h in CNE-2 and CNE-2R cells. Scale bars: 10 μm. (B) Statistical analysis histogram of LC3-labeled red fluorescence puncta in per cell and over 30 cells were counted in each group. (n = 3; ^**p < 0.01; ^***p < 0.005 compared with control group). (C) Western blotting analysis of LC3-I, LC3-II and P62 levels in CNE-2 and CNE-2R cells treated with the indicated concentrations of DMC for 24 h and 48 h. GAPDH was used as a loading control. (D) Transmission electron microscopy images in CNE-2 and CNE-2R cells after treating with control or DMC (40 μM) for 24 h. Red arrows: autophagosomes. Scale bars: 500 nm. (E, F) Western blotting analysis of LC3-I, LC3-II and P62 levels in CNE-2 and CNE-2R cells after treating with 0.1% DMSO or 20 μM DMC in the absence or presence of 3-MA (5 mM) and CQ (20 μM) for 24 h. GAPDH was used as a loading control. (G) Representative images of LC3-labelled green fluorescence puncta after treating with 0.1% DMSO or 20 μM DMC in the absence or presence of 3-MA (5 mM) and CQ (20 μM) for 24 h in CNE-2 and CNE-2R cells. Scale bars: 10 μm.