Sirt1 sirna

Cells were transfected with control siRNA (sc-37007), Sirt1 siRNA (sc-40987), or Atf5 siRNA (sc-60222) purchased from Santa Cruz Biotechnology, in combination with Lipofectamine RNAiMAX Reagent (13778150, Invitrogen) following the manufacturer's protocol 50 (link).

The SIRT1 expression vector pcDNA3.1-SIRT1 and Nrf2 expression vector pcDNA3.1-Nrf2 were purchased from GenePharma (Shanghai, China). The SIRT1 siRNA was purchased from Santa Cruz Biotechnology (sc-40986, Dallas, TX, USA). Cells were seeded in 96-well plates (1 × 10⁴ cells/well) or 6-well plates (1 × 10⁵ cells/well) and transfected with empty or expression vector (1 μg/mL) or siRNA (50 nM) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) at 37 °C according to the manufacturer’s instruction. Forty-eight hours later, Western blot analysis was performed to examine the efficiency of transfection. Then, after transfection for 48 h, cells could be used for the subsequent experiments.

miR-181a-5p inhibitor (5′-ACUCACCGACAGCGUUGAAUGUU-3′) and inhibitor control (5′-UCACAACCUCCUA-GAAAGAGUAGA-3′) were purchased from GenePharma (Shanghai, China). Before transfection, the cells were substituted in an antibiotic-free medium for 24 h, then inhibitor control, miR-181a-5p inhibitor, control-siRNA (cat no. sc-36869; Santa Cruz Biotechnology, Inc., USA), SIRT1-siRNA (cat no. sc-40986; Santa Cruz Biotechnology, Inc.) or miR-181a-5p inhibitor+SIRT1-siRNA were transfected into RAW 264.7 macrophages using Lipofectamin™ 2000 reagent (Invitrogen, USA) following the manufacturer’s instructions. qRT-PCR and western blot analysis were used to measure the efficiency of cell transfection.

Sirt1 siRNA and control siRNA obtained from Santa Cruz were transfected using the Lipofectamine reagent, and the experiments were performed as previously described [24 (link)].

Fibroblasts from healthy subjects were cultured in complete medium without antibiotics for 2 days. Cells were seeded into a six-well plate. Then, 8 μl of LipofectamineTM LTX and 3 μL PLUSTM Reagent (Life Technologies, Carlsbad, CA, USA) were diluted in 90 μL of culture medium. Subsequently, 12 μL SIRT1 siRNA (siRNA for SIRT1-sc-40986- from Santa Cruz Biotechnology) was mixed with the medium containing Lipofectamine together with PLUS reagent and incubated for 30 min at RT for complex formation. Finally, cells were incubated with a final SIRT1 siRNA concentration of 100 nM. After 48 h, SIRT1 protein expression was determined by Western blot. To study the possible role of SIRT1 in the oxidative-mediated cell injury, untreated and SIRT1 RNAi-treated fibroblasts obtained from healthy subjects were challenged for 3 h with 500 μM H₂O₂. ROS production, lipid peroxidation, caspase-3 activity and mitochondrial membrane polarization were evaluated by confocal microscope analysis.

Control siRNA and SIRT1 siRNA were purchased from Santa Cruz (Dallas, TX, USA). PX-478 and SRT1720 were purchased from Apexbio (Houston, TX, USA), tunicamycin from Tocris (Bristol, UK) and EX-527 from RayBiotech (Peachtree Corners, GA, USA). Plasma triglyceride levels were analyzed using a commercial kit (Spinreact SA, St. Esteve d’en Bas, Spain).