Fig. 7. LYN-1604 induces ATG5-dependent autophagy via the ULK complex. (A) LYN-1604 can obviously up-regulate the expression of p-ULK1. The expression of p-ULK1 (Ser317) was detected by immunocytochemistry. Red: TRITC-anti-p-ULK1; blue: DAPI. Scale bar = 10 μm. (B) The expressions of the ULK complex were detected by western blot analysis. β-Actin was measured as loading control. (C) MDA-MB-231 cells were transfected with control or ULK1 siRNA, followed by treatment with 2.0 μM LYN-1604 for 24 h. Then, the expression of LC3B puncta was detected by immunocytochemistry. Green: anti-LC3B; blue: DAPI. Scale bar = 20 μm. (D and E) MDA-MB-231 cells were transfected with control or ULK1 siRNA, followed by treatment with 2.0 μM LYN-1604 for 24 h. Then, the expression levels of ULK complex (D), as well as p62 and LC3 (E) were determined by western blot analysis. β-Actin was measured as the loading control. (F) MDA-MB-231 cells were transfected with control or ATG5 siRNA, followed by treatment with 2.0 μM LYN-1604 for 24 h. Then, the expression levels of ATG5, ULK1, p-ULK1 (ser317, ser757), p62 and LC3 were determined by western blot analysis. β-Actin was measured as the loading control. (G) MDA-MB-231 cells were transfected with control, ATG5 or ULK1 siRNA, respectively. After treatment with 2.0 μM LYN-1604 for 24 h, cell viabilities were measured by MTT assay.