Sirna

To knockdown hCINAP or HIF-1α in AC16 cells, the following siRNAs (all purchased from Sangon Biotech Co., Ltd.) were used: siRNA-hCINAP (5′-GAGAGAAGGUGGAGUUAUU-3′), siRNA-HIF-1α (5′-AAGCAUUUCUCUCAUUUCCUCAUGG-3′) and siRNA-ctrl (scrambled siRNAs) (5′-GACUACUGGUCGUUGAACU-3′). AC16 cells (1×10⁵ cells/well) were transfected with 30 nM siRNA-ctrl, 30 nM siRNA-hCINAP, 1 µg empty vector or 1 µg hCINAP-Flag vector at room temperature using Effectene Transfection Reagent (Qiagen GmbH) according to the manufacturer's protocol and flow experiments were performed 48 h after transfection. To induce LDHA activation, AC16 cells were transfected with 50 or 100 ng vector of hCINAP-Flag at room temperature using Effectene Transfection Reagent (Qiagen GmbH) according to the manufacturer's protocol.
For co-immunoprecipitation experiments, total extracts of AC16 cells were lysed using IP lysis buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.4, 5% glycerol, 1% NP-40, 1 mM EDTA). Lysate (1 ml) was incubated overnight at 4°C with 1 µg hCINAP antibody. Protein A/G-Sepharose (60 µl) was added to samples and incubated at room temperature for 4 h on a rotary shaker (300 × g). The pellets were washed three times with 1 ml lysis buffer and eluted by boiling with 200 µl SDS-PAGE loading buffer. Samples were analyzed via western blotting as described above.

Transfection of siRNA into HUVECs was performed as previously described (25 (link)). The siRNAs (100 nM; Sangon Biotech Co, Ltd.) used in the present study were: p47phox siRNA, 5'-GGACCCAGAACCCAACUAUGCAGGT-3' and 5'-ACCUGCAUAGUUGGGUUCUGGGUCCUC-3'; p22phox siRNA, 5'-GAAGGGCUCCACCAUGGAGTT-3' and 5'-UCCAUGGUGGAGCCCUUCTT-3'; siRNA-scramble, 5'-GCUGCAGTAUGAGGAG-3' and 5'-CGACGC CATUCCGTAGC-3'. Transfections were carried out using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, where subsequent experimentation was performed 48 h after transfection. The mRNA expression of p47phox and p22phox in transfected cells was determined by reverse transcription-quantitative PCR (RT-qPCR).

PBMCs were seeded in 24-well culture plates at a density of 1 × 10⁵ cells per well in 500μL antibiotic-free culture medium. PBMCs were transfected with small interfering RNA (siRNA; Sangon Biotech, China) for the inhibition of JUND expression, and a negative control siRNA (NC) was used as the negative control. The mixture of dilute Lipofectamin2000 (Thermo Fisher Scientific, USA) and siRNA was added to the culture plate according to the instruction. Plated PBMCs were transfected with JUND siRNA at a final concentration of 50 nM. Following this, the plates were then cultivated in a CO₂ incubator at 37°C for 8 h. Post incubation, the transfected PBMCs were submitted to centrifugation to remove any culture medium that could contain residual siRNA, and then cultured in complete culture medium. Forty-eight hours post-transfection, the transfected PBMCs were used for co-culture with HRMECs. Verification of JUND knockdown was achieved via Western blotting.

Low-expressed lncRNA NEAT1 was obtained in SRA01/04 cells using small interfering RNA (siRNA) against NEAT1 (5'-GAGCAATGACCCCGGTGACG-3') and a non-targeting siRNA as a normal control (NC, 5'-TAGATACCCCCAGGCCTACC-3'), which were synthesized by Sangon Biotech (Shanghai, China). SRA01/04 cells were transfected with si-NEAT1 or si-NC using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CA, USA), according to the manufacturer's instructions. After that, SRA01/04 cells were treated with H₂O₂ (300 µM).