Si sirt3

First, we treated the NPCs with DHJSD (300 μg/mL) for 24 h before administrating IL-1β to investigate the effect of DHJSD on NPCs. After that, we pretreated NPCs with DHJSD (300 μg/mL) alone or combined with cyclosporin A (1 μM) for 24 h before IL-1β administration. To explore how miR-494 affected NPCs, we designed a miR-494 inhibitor and mimic and their negative control and synthesized them through GenePharma (Shanghai, China). They were then transfected into NPCs using lipofectamine 2000 before IL-1β administration. To knock down SIRT3 expression, scrambled siRNA (siScr) and short interfering (si) RNA targeting SIRT3 (si-sirt3) were designed and bought from GenePharma (Shanghai, China). The NPCs were co-transfected by adopting miR-494 suppressor (150 nM) and si-sirt3 (100 nM) for 48 h using lipofectamine 2000. Finally, NPCs were either treated with DHJSD alone or pretransfected with miR-494 mimic (50 nM) or si-sirt3 (100 nM) for 24 h to explore the role of mir-494/SIRT3/mitophagy signal axis on DHJSD activity.

Sirt3 (si-Sirt3; GenePharma, Shanghai, China) or negative control- (si-NC; Santa Cruz Biotechnology, Santa Cruz, CA, USA) specific small interfering RNA (siRNA) was transfected into HK-2 cells. The efficiency of silencing Sirt3 after transfection was determined by western blotting experiments.

The MFF-overexpressing vectors (pcDNA3.1-MFF), small interfering RNA (siRNA) targeting to MFF, SIRT1 and SIRT3 (si-MFF, si-SIRT1, si-SIRT3), miR-340-5p mimics, and miR-340-5p inhibitor as well as their corresponding negative controls (pcDNA3.1-NC, si-NC, inhibitor NC, mimics NC) were synthesized and purchased from Genepharma (Shanghai, China). PAMSCs were maintained in a 96-well plate and the transfection experiments were performed by using Lipofectamine™ 3000 (Invitrogen, CA, USA). After 48 h, the transfection efficiency was measured using qRT-PCR method.