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. 2014 Dec 26;9(12):e116165. doi: 10.1371/journal.pone.0116165

Figure 4. ULK1 regulates SIRT1 protein expression which is independent of autophagy.

Figure 4

(A) HEK293 cells were transfected with pcDNA or ULK1 plasmid for 48 h; (B) HEK293 cells were transfected with pcDNA or ULK1 plasmid for 48 h. SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were treated with rapamycin (2 µM) for the indicated time; (D) U20S cells which expressed GFP-LC3 reporter were treated with rapamycin (2 µM) or NONOate (50 µM) for 12 h; GFP-LC3 imaging was captured using fluorescent microscope according to instructions of the manufacturer, EMD Millipore; (E) MEF from the Ulk1 −/−, Ulk2 −/−, Ulk1/2 −/−, and WT mice were treated with bafilomycin A1 (10 nM) for 4 h; (F) HUVECs were treated with NONOate (50 µM) for the indicated time; (G) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (H) HUVECs were treated with A23187 (1 µM) for the indicated time. The western blots are representative of three independent experiments. *represent p<0.05 vs control (n = 3). NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, adenovirus; Baf A1, Bafilomycin A1.