Lv shnc

The full-length FOXK1 cDNA (GenBank accession number NM_001037165) was amplified and subcloned to into pCDNA3.1 expressing vector (Invitrogen, Carlsbad, CA). The expression level of FOXK1 was then examined by western blot. Empty vector- transfected cells were used as a control.
Small interfering RNA (siRNA) targeting FOXK1 and negative control was specifically synthesized by GenePharma (Shanghai, China). Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) was used to transfect siRNAs into cells according to the manufacturer's protocol. The siRNA sequences were as follows: siFOXK1 sense strand, 5′-GCAUGGGCCUGUCCAGCUUT−3′; the scrambled (src) siRNA 5′-TTCTCCGAACGTGTCACGT-3. The siRNA-transfected cells were harvested in the medium for 2 days after transfection and then used for subsequent studies.
Lentiviral vectors containing human FOXK1 short-hairpin RNA (Lv-shFOXK1) and scrambled non-targeting shRNA (Lv-shNC) which was served as negative control were provided by Genechem Company (Shanghai, China). The guiding strand sequences of Lv-shRNA were as follows: shFOXK1, 5′- CCTCTCTCTTAACCGCTACTT-3′; shNC, 5′- TTCTCCGAACGTGTCACGT-3′; Cells were infected with indicated lentivirus at an multiplicity of infection (MOI) of 20 for 48 h and then selected with puromycin (1.5 μg/mL) for 7 days. The expression level of FOXK1 in the infected cells was validated by western blot analysis.

MNK3 cells were cultured in a cell medium composed of DMEM, 10% FBS, and penicillin/streptomycin. For the construction of the SIRT3 knockdown cell line, MNK3 cells were co-cultured with SIRT3 knockdown lentivirus (LV-shSIRT3) or blank vector (LV-shNC) (Shanghai Genechem Co., Ltd, Shanghai, China.) for 12 h, and then the culture medium was replaced. The lentivirus-infected cells were screened in a culture medium containing puromycin for purification. To detect the effect of DHM and palmitic acid (PA) on MNK3 cells, cells were pretreated with DHM for 4 h followed by PA (0.2 mM) for another 12 h. To detect the involvement of AMPK, SIRT3, or STAT3 in DHM-induced benefits on MNK3 cells, cells were pretreated with inhibitors of SIRT3 (3-TYP, 50 μM; MCE, Newark, USA), AMPK (Dorsomorphin, 10 μM; MCE) or STAT3 (Static, 5 μM; Selleck Chemicals, TX, USA) for 2 h prior to the treatment of DHM and PA.

Small interference RNA against circ_0005962 and negative control were synthesized by Sangon Biotech (Shanghai, China). The mimics of miR-382-5p (miR-382-5p), the inhibitor of miR-382-5p (anti-miR-382-5p), and respective negative control (NC or anti-NC) were purchased from Ribobio (Shanghai, China). The overexpression of circ_0005962 (circ_0005962) was performed as the previous study described [24 (link)] and constructed by Genechem (Shanghai, China), while the specific plasmid without the circ_0005962 cDNA was served as a control (circ-NC). The vector pcDNA3.1-PDK4 for the overexpression of PDK4 and the control pcDNA empty vector (vector) were constructed by Sangon Biotech. Lentiviral vector (Lenti-short hairpin) for stable NEAT1 downregulation (Lv-sh-circ_0005962) and the corresponding negative control (Lv-sh-NC) were obtained from Genechem. Cell transfection was conducted using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Transfection efficiency and the following experiments were implemented after 48 h of transfection.

The lentivirus-mediated shRNA for Rgs4 (LV-shRGS4) (5′ CACC GGGA GGTT CACA TCCT AAAC GAAT TTAG GATG TGAA CCTCCC3′) and the lentivirus carrying scrambled shRNA (LV-shNC) (5′GTTC TCCG AACG TGTC ACGT3′) as negative control were synthesized by Genechem (Shanghai, China). The lentivirus-mediated Rgs4 (LV-RGS4) was used to overexpress Rgs4. Briefly, mice were anaesthetized, a thoracotomy was performed through the fourth intercostal space. The ascending aortic artery and the main pulmonary artery were clamped. The lentivirus was injected (2.5 × 10⁷ TU·mL⁻¹ at a volume of 100 μL) through the tip of the heart into the left ventricular cavity. The arteries were occluded for 10 s after lentivirus injection [21 (link)]. Myocardial infarction operation was started 7 days after lentivirus injection. The treatment process of mice was shown in Table 1.Table 1

The treatment process of mice

	0 day	7 day	10 day	35 day
Part I	Lentivirus injection (LV-shRGS4/NC)	MI surgery	–	Collected heart tissue
Part II	Lentivirus injection (LV-RGS4/NC)	MI surgery	Choline treatment	Collected heart tissue

Three siRNAs targeting sclerostin (SOST, Gene ID: 74499) (siSOST) and negative control RNAs (siNC) were synthesized by Sangon (Shanghai, China). At 16–18 h of seeding, the BMSCs approximately at 80% confluence were transient transfected with siRNAs by Lipofectamine3000 (L3000015, Invitrogen, CA, USA) according to the manufacturer’s protocols.
The LV-Fto-RNAi (LV-shFTO) and LV-negative control (LV-shNC) were constructed by Genechem (Shanghai, China). BMSCs reached 30% confluent were infected with lentivirals by HitransG with MOL 10 according to the manufacturer’s instructions.
MC3T3 cells were chosen to establish stable FTO knockdown models, that were infected with LV-shFTO (Mol = 5) and were selected using puromycin (2 μg/ml). Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot (WB) were used to identify the efficiency of FTO knockdown. The primer sequences for PCR are provided in Additional file 1. The targeted sequences for siRNAs and shFTO are listed in Additional file 2.
Full details of the materials and methods are provided in Additional file 3.