Oe nc

For in vitro overexpression studies, HeLa and SiHa cells were selected for they showed the lowest expression of hsa_circ_0107593. For knockdown experiments, CaSki and ME180 were selected for they showed the highest expression of hsa_circ_0107593. A plasmid overexpressing hsa_circ_0107593 was transfected into HeLa and SiHa cells. siRNAs targeting hsa_circ_0107593 were transfected into CaSki and ME180 cells. Three siRNAs (siRNA-1, siRNA-2, and siRNA-3) and corresponding negative control siRNA (si-NC) were purchased from RiboBio (Guangzhou, China). The siRNA with highest knockdown efficiency was selected for all subsequent experiments. The overexpressing plasmid and its corresponding negative control plasmid (OE-NC) were purchased from TSINGKE Biotech Ltd. (Beijing, China). Cells were harvested 48 h post-transfection, then transfection efficiency was checked by qRT-PCR. For dual-luciferase reporter assay, hsa-miR-20a/93/106b-5p mimics, as well as their respective negative control (NC mimics) were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Cell transfection was performed with Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, USA) following the manufacturer’s instructions.

The plasmid encoding the transcript of CXCL2 (oeCXCL2) and the negative control (oeNC) was synthesized by Tsingke (Nanjing, China). The siRNA targeted CXCL2 (siCXCL2) and the negative control (siNC) were purchased from GemmaPharma (Shanghai, China). Cells were transfected by Lipofectamine 3000 Transfection Reagent (L3000150, Invitrogen) for 24–48 h. The CXCL2 siRNA sequence was described in the previous study [22 (link)].

Ad-Slc40a1 and Ad-Ctrl constructs were generated by Hanbio Biotechnology (China) using adenoviral vectors carrying Slc40a1. Ad-shSlc40a1 and Ad-shCtrl were provided by GeneChem Technology (Shanghai, China), in the form of adenoviral vectors carrying shRNA-Slc40a1.Protein expression was observed 48 hours after viral infection. The plasmid containing Steap4 and the negative control plasmid (OE-NC) were created and produced by Tsingke Biotech (China). Cardiomyocytes were transfected with the plasmid using Lipofectamine 3000 from Invitrogen (USA). Protein expression was measured 72 hours after transfection. The shRNA target sequences are shown in Table S3.