Lipofectamine 3000

BNIP3-overexpression plasmid and FUNDC1-overexpression plasmid were purchased from GeneCopoeia (United States). Briefly, human BNIP3 cDNA was cloned from NM_004052.4, mouse BNIP3 cDNA from NM_00976, human FUNDC1 from NM_173794, and mouse FUNDC1 cDNA from NM_028058. The resultant fragments were inserted into the OmickLink^TM Expression Vector pEZ-M02. Cultured 661W photoreceptors, Müller cells, and HUVECs were plated (1 × 10⁶ cell/6 well) in DMEM + FBS. The medium was then exchanged for Opti-MEM (Gibco) with Lipofectamine and 5 μg of the BNIP3- and FUNDC1-overexpression plasmids for 48 h using Lipofectamine 3000 (GLPBIO, United States, GK20006) under normal culture conditions. The HIF-1α inhibitor LW6 was purchased from (GLPBIO, United States, GC32724) and dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM for cell application. Similarly, chloroquine (CQ) (GLPBIO, United States, G6423) was dissolved in DMSO to a final concentration of 10 mM. All these solutions were stored at −20°C before use. Subgroups of cells were incubated with 0.625 mg/mL bevacizumab (Avastin^®, Roche, Switzerland), 0.5 mg/mL aflibercept (Eylea^®, Bayer, Germany), or 0.125 mg/mL ranibizumab (Lucentis^®, Novartis, Switzerland) as indicated. When combined with bevacizumab, LW6 and CQ were used at 25 μM.

The interfering RNA (siRNA- PUS7) was purchased from Genepharma Corporation (Jiangsu, China), and its sequence was 5’-GCUAGGGAAUUUCAGCUAUTT-3’. Genepharma was employed to design a coding sequence for the human PUS7 overexpression plasmid, which was constructed into the pcDNA3.1 vector. According to the manufacturer’s protocol of Lipofectamine 3000 (Glpbio, Montclair, CA, USA), cells were transfected with siRNA or plasmids when inoculated in six-well plates for 8–12 h, and PUS7 expression was measured by western blot 48 h after cells were transfected.

Negative control siRNA (siNC) contrast and siRNA were synthesized by GenePharma. The lipofectamine 3000 (No. GK20006) was obtained from Glpbio Technology Company. The template of siRNA for subsequent interference was determined as follows: siNur77: 5′‐GGU GAU UGG AUU GAC AAC‐ATT‐3′; siNC: 5′‐UUC UCC GAA CGU GUC ACG UTT‐3′. RAW264.7 cells were transfected with 100 pmol siNur77 or siNC in lipofectamine 3000 according to the manufacturer's recommendation. Six hours later, the medium was replaced by DMEM + 10% FBS, cultured for an additional 72 h, and then subjected to the following treatments.
Following cell transfection with siNur77, the treatments for different cell groups were as follows: (1) siNC, transfection with siNC only; (2) siNC + Dex, siNC transfection and Dex treatment; (3) LPS + siNC, siNC transfection with LPS treatment; (4) LPS + Dex + siNC, siNC transfection with Dex and LPS treatment; (5) siNur77, transfection with siNur77 only; (6) siNur77 +  Dex, siNur77 transfection and Dex treatment; (7) LPS +  siNur77, siNur77 transfection with LPS treatment; (8) LPS + Dex + siNur77, siNur77 transfection with Dex and LPS treatment. The concentrations of Dex and LPS to be used were selected as previously described.²⁵, ²⁶, ²⁷