Sirt1 sirna

To silence SIRT1, HepG2 cells were transfected with siRNA against human SIRT1 (33–100 nM) using Lipofectamine 2000 according to the manufacturer’s guidelines. The siRNA sequence was synthesized by Sangon Biotech (Shanghai, China) (SIRT1 siRNA: sense, 5′-UAGGUGCCCAGCUGAUGAAUU-3′, anti-sense, 5′-UUCAUCAGCUGGGCACCUAUU-3′; negative control siRNA: sense, 5′-UUCUCCGAACGUGUCACGUUU-3′, anti-sense, 5′-ACGUGACACGUUCGGAGAAUU-3′).The shRNA sequence (SIRT1 shRNA, 5′-GATCCTAGGTGCCCAGCTGATGAGTTCAAGAGACTCATCAGCTGGGCACCTATTTTTG-3′ or control shRNA, 5′-GATCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3′) was synthesized by Sangon Biotech and inserted into the pGreenPuro vector (SBI). Recombinant lentiviruses encoding mouse SIRT1 shRNA (LV-shSIRT1) or negative control shRNA (LV-shCtrl) were treated as described previously34 (link).

The specific siRNA sequences, including Sirt1-siRNA, Nrf2-siRNA and the negative control, were synthesized by Sangon Biotech (Shanghai, China). The sequences of Sirt1-siRNA were as follows: sense: 5′-CCAGUAGCACUAAUUCCAATT-3′, antisense: 5′-UUGGAAUUAGUGCUACUGGTT-3′. The sequences of Nrf2-siRNA were as below: sense: 5′-GAGGAUGGGAAACCUUACUTT-3′, antisense: 5′-AGUAAGGUUUCCCAUCCUCTT-3′. Appropriate siRNA was transiently transfected into GMCs with lipofectamine^® RNAiMAX (Invitrogen, United States). After 48 h incubation, the transfection medium was replaced with fresh serum-free DMEM for another 12 h. After various treatments, the cells were harvested for WB.

SIRT1-siRNA was designed and synthesized by Sangon Biotech. The SIRT1-siRNA sequences were as follows
Forward: 5’-GCGGGAAUCCAAAGGAUAAUUTT-3’.
Reverse: 5’-AAUUAUCCUUUGGAUUCCCGCTT-3’
Control-siRNA
Forward: 5’-UUCUCCGAACGUGUCACGUTT-3’
Reverse: 5’-ACGUGACACGUUCGGAGAATT-3’.
KGN cells were cultured in a 6-well plate until they reached 70-80% confluence. Then, siRNA, serum-free culture medium, and transfection reagent Lipo8000™ were diluted in the appropriate proportions. The siRNA mixture was added to the cells in the 6-well plate. The cells were transfected for two days, and then SIRT1 protein expression was assessed.