Ribofect cp

Cells were seeded on 6-well plates at 50-60% confluence before transfection. siRNAs for Mfn2 (siMfn2) were transfected into cardiac fibroblasts for 24 h using the transfection reagent riboFECT™ CP (RiboBio™, China) before stimulation with NAC (Selleck, S1623) for 2 h and then TGF-β1 for 24 h. Individual siRNAs (100 nM, RiboBio™, China), riboFECT™ CP buffer, riboFECT™ CP reagent, and DMEM were mixed, incubated at room temperature for 10-15 min, and then added to the cell cultures. All siRNA sequences are shown in Table 1.

Cells grown to 40-50% confluence were transferred to 6-well plates. They were transfected using transfection reagent riboFECT™ CP (RiboBio™, China). siRNA targeting Sig1R (si-Sig1R) was transfected into NRCFs for 24 hours then treated with TGF-β1 for 48 hours. Individual siRNAs (100 nM, RiboBio™, China), ribo-FECT™ CP reagent and buffer, and DMEM were combined and then incubated for 15 minutes at room temperature. The experiment was divided into four groups: siNC group, siNC+T group, si-Sig1R group, and si-Sig1R+T group.

BALB/c mice of 5–7 days age (Animal Center, Guangxi Medical University, China) were used to extract primary osteoblasts via enzyme digestion (Type II Collagenase, Gibco, CA, USA). Primary osteoblasts were identified using alkaline phosphatase staining (Beyotime, Shanghai, China). Vascular endothelial cells were obtained from Aolu Company (Shanghai, China). These were cultured in MEMα medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA) and 100 U/ml each of penicillin and streptomycin (Gibco, CA, USA). Cells were maintained in a thermostatic incubator with 5% CO₂ at 37°C. In the coculture system, osteoblasts were seeded in the upper chamber of a Transwell chamber (Corning, USA) at a density of 2.0 × 105 cells/ml and endothelial cells were inoculated in 6-well plates (Corning, USA) at a density of 1.0 × 105 cells/ml. The Transwell chambers were placed into plates after both cell types reached adherent growth densities of 50%. miR-139-5p mimic and inhibitor with their negative controls were synthesized using riboFECT CP (RiboBio, Guangzhou, China) and transfected into endothelial cells using riboFECT CP reagents, following the manufacturer's instructions.