Lipofectamine ltx and plus reagent kit

HepG2 was transiently transfected with Beclin siRNA and control siRNA using Lipofectamine LTX Plus reagent kit according to manufacturer’s protocol (Invitrogen, USA). 0.35 x 10⁶cells per well were seeded in a six-well tissue culture plate containing 2ml antibiotic-free normal growth medium supplemented with FBS and the cells were incubated to attain 60% confluency and transfected using the Lipofectamine LTX and Plus Reagent Kit (Invitrogen, USA) according to manufacturer’s protocol. The silencing of Beclin expression was confirmed by Western blotting with anti-beclin-1 and the transfection efficiency was standardized at 50-60h before the drug treatment and Utt-B was treated for 8-12h.

The pGFP-mRFP-LC3B (ptf-LC3) vector was purchased from Addgene (21074). Briefly, the plasmid present in the bacterial pellet was isolated according to the manufacturer’s instruction (GenElute™ Plasmid Miniprep Kit- Sigma-Aldrich). HepG2 cells were transfected transiently with tandem repeats of GFP-RFP tagged LC3 (ptfLC3) using the Lipofectamine LTX and Plus Reagent Kit (Invitrogen, USA) according to the manufacturer’s protocol. HepG2 cells were seeded at a density of 0.25x 10⁶ in a coverslip and were treated with Utt-B for 24h after transfection, examined, and photographed. DAPI staining was performed by 10 min incubation with 1µg/ml DAPI in HepG2 cells treated with mentioned concentrations of Utt-B and Cqn and viewed under the confocal microscope.