Fig 6. ANKRD49-regulated autophagy induced by serum-starved GC-1 cells is dependent on the NF-κB pathway.
(A, B) GC-1/ANKRD49-Flag stable cells were pre-treated with 50 μm PDTC or 10 μm BAY 11–7082 for 2 h prior to treatment with serum-free media for another 24 h, and the cell lysates were prepared and blotted with the indicated antibodies. (C) GC-1/ANKRD49-Flag stable cells were transfected with mouse NF-κB/p65 siRNA and a negative control for 24 h and incubated in serum-free media for another 24 h. Cell lysates were prepared and blotted with the indicated antibodies. β-actin served as a loading control. One representative of three independent experiments is shown. The quantitative results are presented as the ratio of LC3-II to LC3-I (n = 3). **p < 0.01 indicates significant difference between groups as shown. (D) GC-1/ANKRD49-Flag cells and control cells were transfected with GFP-LC3 for 24 h, followed by serum starvation culture with or without PDTC and BAY 11–7082 for another 24 h. Punctated GFP-LC3-Ⅱ was observed by a Confocal Laser Scanning Microscopy (left) and a punctuated pattern was shown (right), indicating appearance of autophagy. Scale bar represents 100 μm. **p < 0.01 indicates significant difference between groups as shown.