Lentiviral construct

CMs or CMVECs were transfected at approximately 80% confluence using a lentiviral construct (HANBIO, China) according to the manufacturer's protocol. Some of those lentiviral constructs were empty (LV), and some carried synthesized circHIPK3 (LV-circHIPK3), small interfering RNAs (Sigma) targeting circHIPK3 (LV-sicircHIPK3), or linear HIPK3 (LV-silineHIPK3). In addition, an miR-29a inhibitor, miR-29a mimics, and negative controls were all synthesized by RiboBio (Guangzhou, China) and transfected into CMVECs by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. CMVEC functions and gene expression were evaluated 48 h after transfection. The sequence information is listed in Table 1.

The synthesized siR-PTEN (siR-PTEN) and scramble (GeneCopoeia, MD) were transfected into C-kit⁺ CSCs using a lentiviral construct (HANBIO, China) according to the manufacturer’s instructions. Briefly, The lentiviral vector expressing PTEN(siR-PTEN) or PTEN negative control (siR-PTEN-NC)were constructed by inserting the siR-PTEN gene or siR-PTEN-NC into a Lv-EGFP vector using BamHI (FD0054) and EcoRI (N41890) restriction sites, all obtained from Invitrogen (Thermo Fisher Scientific). The lentiviral particles were prepared using a calcium phosphate method.The C-kit⁺ CSCs (1× 10⁵ per well) were plated into 6-well plates and then treated with siR-PTEN and siR-PTEN-NC in the presence of 2 μg/ml polybrene (Sigma-Aldrich) at a multiplicity of infection of 50 MOI for 48 h. The siR-PTEN knockdown efficiency was confirmed by Western blotting and RT-qPCR.

CircHIPK3, small interfering RNAs (siRNAs) against circHIPK3, the linear HIPK3 gene, and siRNAs against HIKP3 were transfected into CMs or cardiac endothelial cells using a lentiviral construct according to the manufacturer's protocol (Hanbio, China) and performed as previously published [11 (link)]. In addition, miR-29a mimic, inhibitor, or their corresponding negative controls (RiboBio, China) were transfected into cardiac endothelial cells with Lipofectamine 3000 (Invitrogen, USA) according to the kit's instructions. At 48 h after transfection, qPCR was used to evaluate the gene expression in cardiac endothelial cells.