plasmid dna mix lipofectamine ltx reagent  (Thermo Fisher)

Journal: Cell Death & Disease

Article Title: A new molecular mechanism underlying the EGCG-mediated autophagic modulation of AFP in HepG2 cells

doi: 10.1038/cddis.2017.563

Figure Lengend Snippet: EGCG induced intracellular AFP degradation through autophagy in HepG 2 cells. ( a ) HepG 2 cells were stimulated with 25 and 50 μ M EGCG for 24 h. The 18-kDa cytosolic LC3-I and 16-kDa lipidated autophagosome-bound LC3-II was analyzed by the western blot method. ( b ) HepG 2 cells were stimulated with 25 and 50 μ M EGCG for 24 h, and then stained with acidotropic MDC dye to visualize autophagosomes (yellow arrow: autophagosomes) (× 40). ( c ) GFP-transfected HepG 2 cells were stimulated with 50 μ M EGCG for 24 h. Cytoplasmic aggregated AFP were partly colocalized with LC3-containing vesicles (as shown by yellow arrows) (× 60). ( d ) EGCG enhanced intracellular AFP level in the presence of 3-MA. HepG 2 cells were stimulated with 50 μ M EGCG for 16 and 24 h, and 10 mM 3-MA was added at 4 h before EGCG treatment. Western blot analysis was conducted to determine extracellular, intracellular LC3, and AFP level with reference to β -actin

Article Snippet: The GFP-LC3 plasmid/Lipofectamine mixture (5.0 ml, containing 2.5 μ g plasmid DNA) was added to the HepG 2 cell culture, and incubated for 12–16 h. Afterwards, HepG 2 cells stably expressing GFP-LC3 were selected and maintained in DMEM/10%FBS/2 mM glutamine supplemented with geneticin (600 μ g/ml; Gibco, China).

Techniques: Western Blot, Staining, Transfection