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. 2016 Aug 17;12(11):2009–2025. doi: 10.1080/15548627.2016.1215382

Figure 2.

Figure 2.

Inhibition of hypothalamic AMPK suppresses autophagy induced by low glucose availability. (A) N41 and N43/5 cells were treated with 2DG (20 mM) or exposed to glucose-free medium (0 mM glucose) with or without compound C (CC: 16 μM) for 4 h. The levels of p-AMPK (Thr172), total AMPK, p-ACAC (Ser79), total ACAC, p-ULK1 (Ser555), total ULK1, p-RPTOR (Ser792), total RPTOR, LC3, and GAPDH were determined by immunoblotting. (B) Treatment with lysosomal protease inhibitors, E64d (10 μg/ml) and pepstatin A (PepA: 10 μg/ml), for 4 h in compound C-treated N41 and N43/5 cells confirmed increased autophagy flux induced by low glucose availability. (C) N41 and N43/5 cells transiently expressing mRFP-GFP-LC3 were treated with 2DG or glucose-free medium with or without CC for 4 h. The bottom graphs indicate relative fluorescence intensity (RFI) of RFP and GFP along a white line in the upper enlarged images. The Y-axis shows RFI and the X-axis shows relative distance. Scale bars: 10 μm. (D) Average number of mRFP-GFP-LC3 puncta per cell. At least 30 cells were counted per condition. Red bar and symbols refer to red puncta, yellow bar and symbols to yellow puncta; and white bar and black symbols to total puncta (***, p < 0.001 for 25 mM glucose vs. 2DG or glucose-free medium; ###, p < 0.001 for vehicle vs. CC in 2DG-treated groups; †††, p < 0.001 for vehicle vs. CC in groups exposed to glucose-free medium). (E) N41 and N43/5 cells infected with lentiviruses containing control or both AMPK (against α1 and α2) shRNAs were exposed to low glucose conditions for 4 h, and the levels of p-AMPK (Thr172), total AMPK, p-ACAC (Ser79), total ACAC, p-ULK1 (Ser555), total ULK1, p-RPTOR (Ser792), total RPTOR, LC3, and GAPDH were detected by immunoblotting. p-AMPK, p-ACACB (upper band), p-ULK1, and p-RPTOR were normalized to corresponding total proteins. Protein levels of p-AMPK (in panel D), total AMPK, and LC3-II were normalized to GAPDH. Numbers under blots indicate relative quantitative mean values of independent replicates (A and D: n = 2, B: n = 3).