Beclin 1 sirna

Lipofectamine 2000 reagent and the Invitrogen protocol were used to introduce Beclin-1 siRNA (Sigma-Aldrich; seq1 #SASI_Hs02_00336256) or a scramble control siRNA sequence (Sigma-Aldrich; #SIC001) into Ishikawa cells. Cells were then incubated for 48 h prior to metformin treatment (0, 5, or 10 mM).

RAW264.7 macrophages (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin under a humidified condition of 5% CO₂ at 37°C.
The transient transfection of small interfering RNA (siRNA) using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) was performed according to the manufacturer's protocol. In brief, the siRNAs specific for PERK, CHOP and beclin‐1 (200 pmol) or control siRNA oligomers (200 pmol) were dissolved in 250 μL of Opti‐MEM I reduced serum medium (Invitrogen), and then gently mixed with 5 μL of Lipofectamine 2000 (Invitrogen) pre‐diluted in 250 μL of Opti‐MEM. After 20 min incubation at room temperature, the transfection complexes were added to cells grown in 6‐well plates in a final volume of 2.5 ml of medium and incubated for 6 h. Thereafter, the transfection medium was replaced with normal medium. After transfection for 48 h, the cells were exposed to gly‐HDL (100 mg/L) for 24 h. PERK siRNA (5′‐GUAAUGGUGCACUUUCAAUdTdT‐3′), CHOP siRNA (5′‐CUCUCCAGAUUC CAGUCAGdTdT‐3′) and beclin‐1 siRNA (5′‐CAAGUUCAUGCUGACCAAUdTdT‐3′) were obtained from Sigma‐Aldrich. The efficiency of siRNA‐silenced genes was validated by Western blot analysis.