Gfp lc3 vector

When autophagy is induced, cytosolic LC3 is cleaved by hydrolysis to a shorter peptide (LC3 II) that is located on the membrane of the autophagosome; therefore, the expression levels of LC3 II may be used to estimate the level of autophagy. In order to estimate the location and processing of LC3-II, assessment of LC3-GFP puncta is an effective method (16 (link)). Panc-1 and Colo-357 cells were incubated in 6-well plates with DMEM supplemented with 10% FBS at 37°C in an atmosphere containing 5% CO₂. When they had reached 80% confluence, the cells were incubated with Opti-MEM medium (Thermo Fisher Scientific, Inc.), and transfected with a GFP-LC3 vector (GeneChem Co., Ltd., Shanghai, China) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h. Subsequently, the cells were treated with DMSO or 40 µM wogonin for 24 h, then fixed with 4% paraformaldehyde for 15 min, and washed three times with PBS. The cells were imaged under a fluorescence microscope (Nikon TE2000) and LC3-GFP puncta-positive cells were counted (≥5 puncta was considered positive).

HuH7 and HepG2 cells were transfected with GFP-LC3 vector (GeneChem, Shanghai, China) through Lipofectamine 2000 (Invitrogen Life Technology, Carlsbad, CA, USA). After 18 h, cells were treated with drugs for 24 h, using 4% faraformaldehyde to fix for 10 min, washed 3 times with PBS, and then observed under fluorescence microscope (Nikon TE2000; Nikon).