Figure 3.

Overexpression of ATG4B promotes autophagy in ES cells. (A) TC71 cells were transfected with ATG4B expression plasmid or control plasmid, and overexpression of ATG4B was examined by western blotting. (B) Western blot analysis of p62, LC3-I, LC3-II and GAPDH in ATG4B overexpressed TC71 cells following starvation for 2 h. (C) Band intensity of p62 and LC3-II were quantified and normalized to the GAPDH in (B). (D) TC71 cells transfected with ATG4B expression plasmid or control plasmid were inspected under confocal laser microscopy to detect LC3 puncta by immunofluorescence after starvation for 2 h. Magnification, ×400. (E) LC3 puncta number per cell in (D) was quantified using the ImageJ program, and 30–60 cells chosen in random were counted. Data are representative of three independent experiments (mean ± standard deviation). ^***P<0.001 as indicated. ES cells, Ewing sarcoma cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.