Adenoviral vector

To make Ad-KLF5 and Ad-GFP as describing previously [13 (link)]. To infect Hela cells with an Adenoviral vector (Genechem Co., Ltd). To achieve RNAi-mediated depletion of Eppk1, we transfected cells with siRNA oligos targeting Eppk1 (si-Eppk1) or negative controls (si-Con) (Genechem Co., Ltd). In this process, we also used the Lipofectamine 2000 reagents (Invitrogen; Thermo Fisher Scientific, Inc.) according to the instructions.

The following were inserted into adenoviral vectors according to the manufacturer's instructions (GeneChem): the full‐length rat lncRNA H19 sequence (Ad‐H19) to ensure H19 expression, a short‐hairpin RNA (Ad‐shH19) directed against lncRNA H19 and the associated negative control oligonucleotides. Recombinant adenoviruses for miR‐22‐3p overexpression (Ad‐miR‐22) and miR‐22‐3p silencing (Ad‐Antagomir‐22), as well as their negative control, were also constructed by GeneChem. The adenovirus encoding KDM3A (Ad‐KDM3A), which has been verified to be efficient in inducing the overexpression of KDM3A in vivo,15 was provided by GeneChem as well, and Ad‐GFP was used as a control.
Five days before the establishment of the AMI model, rats were anaesthetized, and the pericardium was removed through a small left anterior thoracotomy. Then, 100 µL of adenovirus solution was intramyocardially injected into five separate sites of the left ventricle by using a microsyringe. Five days later, the rats that survived were subjected to AMI surgery.

Antibodies against Drp1, p62, LC3, RIP1, RIP3, TSG101, and CD63 were purchased from Abcam (Cambridge, MA, USA). β-actin, tubulin, and COX IV, which were used as references for the total, cytoplasmic, and mitochondrial fractions, were also acquired from Abcam (Cambridge, MA, USA). Antibodies against Phospho-RIP3 (Thr231/Ser232) and Phospho-Drp1 (Ser616) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Aggresome Detection Kit was acquired from Abcam (Cambridge, MA, USA). MitoTracker Deep Red, the ROS 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, dihydroethidium (DHE) assay kit, and MitoSOX detection kit were purchased from Invitrogen (Carlsbad, CA, USA). Ad-mRFP-GFP-LC3 was supplied by Beyotime (Shanghai, China). Adenoviral vectors for Drp1 short hairpin RNA (shRNA) and Drp1 mutations (Drp1-S616D and Drp1-S616A) were generated by Genechem Technology (Shanghai, China). Related activators and inhibitors, including Mitochondrial division inhibitor 1 (Mdivi-1), N-acetylcysteine (NAC), and Necrostatin-1 (Nec-1), were obtained from Selleck (Shanghai, China). The Protein A/G Magnetic Beads IP Kit was purchased from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS) and penicillin/streptomycin were procured by Invitrogen (Carlsbad, CA, USA). All other chemicals were supplied by Sigma unless otherwise specified.