Sirt3 sirna

Normal Primary Human umbilical vein endothelial cells (ATCC® PCS-100-010™) were maintained in endothelial cell medium (ECM; Sciencell, USA) in humidified incubator with 5 % CO₂ and 95 % air at 37 °C until desired cell density was reached. Confluent cells cultured up to 3–7 passages were first starved in 0.5 % fetal bovine serum ECM for 24 h before exposure to AngII (Sigma; 10⁻⁷, 10⁻⁶, 10⁻⁵ mol/L) for another 24 h.
The SIRT3 siRNA used was synthesized by Shanghai GenePharma (Shanghai). Negative control siRNA, which does not target any known gene, was used as a negative control. Cells were transfected with the siRNAs by the Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and examined by western blot analysis. Cells transfected with SIRT3 siRNA were then treatment with or without AngII (10⁻⁶ mol/L) for 24 h.

RNA interference was conducted using the Oligofectamine reagent (Invitrogen). And cultured EPCs were transfected with the 100 nM SIRT3 siRNA or negative control siRNA (GenePharma, Shanghai, China) according to the instruction. Cells had 60%-70% confluence on the day of transfection. After transfecting for 48hours, the knockdown efficiency was tested by western blot.

After starvation for 24 h, lipofectamine 3000 (Thermo Fisher Scientific Inc., Rockford, IL, USA) mixed with SIRT3 siRNA (5′-CCAUCUUUGAACUAGGCUUTT-3′ and 5′-AAGCCUAGUUCAAAGAUGGTT-3′, GenePharma, Shanghai, China) or negative control (NC) siRNA were added into the cell culture medium. After transfecting for 48 h, the SIRT3 protein expression was detected by Western blot to assess the efficiency of siRNA transfection. Then, cardiac fibroblasts were pre-administrated with NaHS (50 μM) for 4 h, followed by incubation with normoxia or hypoxia for another 4 h.

After starvation for 24 h, the mixture of the SIRT3 siRNA or nonspecific control siRNA (NC siRNA) and the Lipofectamine 3000 reagent was added into cardiac fibroblasts. SIRT3 siRNA (5′-CCAUCUUUGAACUAGGCUUTT-3′ and 5′-AAGCCUAGUUCAAAGAUGGTT-3′) or NC siRNA with random sequences was commercially synthesized (GenePharma, Shanghai, China). The expression of SIRT3 mRNA and protein was measured by real-time PCR and western blot, respectively, to evaluate the efficiency of SIRT3 siRNA transfection after 48 h. Some other cells were stimulated with NaHS (50 μM) for 4 h followed by Ang II (100 nM) stimulation for 24 h after siRNA was transfected into the cardiac fibroblasts.

The H9c2 cell line (Cellcook Biotech Co., Ltd., Guangzhou, China) was used for in vitro studies and maintained in DMEM media with 1.0 g/L Glucose (10014CM, Corning, New York, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA). In the following tests, H9c2 cells were cultured in 2% fetal bovine serum (FBS) media for 12 h before treatment. The cells were pretreated with 100 μM apocynin (Sigma, Saint Louis, MO, USA) for 1 h and stimulated with 33 mM high glucose (HG) for 36 h. The cells were divided into the following groups at random: NC, HG, and HG + APO groups.
When cells reached up to 70% confluence, H9c2 cells were seeded into six-well plates and transfected with control siRNA or SIRT3 siRNA (GenePharma, Suzhou, China) for 24 h using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). Then, H9c2 cells were treated with 100 μM apocynin for the next 24 h. Cells were rinsed with PBS after being exposed to apocynin for further measurements.

Septwolves cigarettes (tobacco type of tar: 10 mg, nicotine content: 0.8 mg, carbon monoxide fumes: 12 mg) were from the China Tobacco Fujian industry limited liability company (Xiamen, China). The human bronchial epithelial cell line (BEAS-2B) was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Sirt3, MnSOD, and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Sirt3 siRNA and Sirt3 plasmid were purchased from GenePharma Co. Ltd. (Shanghai, China).