Specific sirna duplexes

The siRNA technique was used to silence subtypes of NMDARs in HUVECs. Cells at a 40–50% confluence were transfected with specific siRNA duplexes (Santa Cruz Biotechnology, CA, USA) using a Transfection Reagent (Zeta Life, San Francisco, USA) following the manufacturer’s instructions. After transfection of control siRNA (sc-37007), NMDAζ1 siRNA (sc-36081), NMDAε1 siRNA (sc-36083), NMDAε2 siRNA (sc-36085), NMDAε3 siRNA (sc-42546), NMDAε4 siRNA (sc-36087), or TLR4 siRNA (sc-40260) (Santa Cruz Biotechnology, Dallas, TX, USA) for 48 h, cells were then collected for western blot, flow cytometry, and mitochondrial functional analysis.

The specific siRNA duplexes targeted against human BRD2, BRD3, or BRD4, siRNA transfection reagent, and reduced-serum transfection medium were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The day before transfection, 4 × 10⁴ cells were seeded in each well of 12-well cell culture plates in RPMI 1640 medium containing 10% FCS without antibiotics and incubated for 24 h. The next day, transfection complexes were prepared using BRD siRNA, siRNA transfection reagent, and transfection medium according to the manufacturer’s instructions and were delivered to cell monolayers in 600 μl fresh media with 50 or 100 nM final concentration of siRNA duplexes. A scrambled siRNA (Santa Cruz Biotechnology) was used as negative control.

The siRNA technique was used to silence specific genes in HUVECs. The cells at 40%-50% confluence were transfected with specific siRNA duplexes (60 nM, Santa Cruz Biotechnology) using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer’s instruction. After 48 h transfection of control siRNA, Sirt1 siRNA or Sirt3 siRNA (Santa Cruz Biotechnology), the cells were incubated with 5.5 mM or 33 mM glucose culture medium in the absence or presence of 3 μM acacetin for 5 days, then collected for western blot analysis. The silencing efficiency of Sirt1 and Sirt3 proteins was significant, which is shown in Supplementary Figure S1.