Silencer
Select Pre-designed siRNA for Nrf2 and negative control siRNA were
obtained from Ambion (Life Technologies, Carlsbad, CA, USA). The target
sequences for the Nrf2 siRNAs were 5′-GAAUGGUCCUAAAACACCATT-3′
(sense) and 5′-UGGUGUUUUAGGACCAUUCTG-3′ (antisense).
Reverse transfections of siRNAs into HepG2 cells were performed using
Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’s protocol. In brief, HepG2 cells were maintained
in antibiotic-free medium. Each siRNA was diluted with Opti-MEM I
Reduced Serum Medium (Gibco, Carlsbad, CA, USA) and added along with
Lipofectamine RNAiMAX to a single well of a 6-well plate. After 20
min of incubation at room temperature, a suspension of 2.5 ×
105 HepG2 cells was added to obtain a 2 nM final siRNA
concentration. After 48 h of incubation, the medium was replaced with
fresh medium with or without OP60, and cells were further incubated
for 24 h. Cells were then harvested for the measurement of GSH and
total RNA preparation. The efficiency of siRNA knockdown of Nrf2 was
determined by real-time PCR.
Select Pre-designed siRNA for Nrf2 and negative control siRNA were
obtained from Ambion (Life Technologies, Carlsbad, CA, USA). The target
sequences for the Nrf2 siRNAs were 5′-GAAUGGUCCUAAAACACCATT-3′
(sense) and 5′-UGGUGUUUUAGGACCAUUCTG-3′ (antisense).
Reverse transfections of siRNAs into HepG2 cells were performed using
Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’s protocol. In brief, HepG2 cells were maintained
in antibiotic-free medium. Each siRNA was diluted with Opti-MEM I
Reduced Serum Medium (Gibco, Carlsbad, CA, USA) and added along with
Lipofectamine RNAiMAX to a single well of a 6-well plate. After 20
min of incubation at room temperature, a suspension of 2.5 ×
105 HepG2 cells was added to obtain a 2 nM final siRNA
concentration. After 48 h of incubation, the medium was replaced with
fresh medium with or without OP60, and cells were further incubated
for 24 h. Cells were then harvested for the measurement of GSH and
total RNA preparation. The efficiency of siRNA knockdown of Nrf2 was
determined by real-time PCR.