Hieff trans reagent

The plasmid for CD36 overexpression was constructed in our previous study and named as pCMV-CD36 (Liang et al. 2018 (link)). For overexpression experiment, cells were transfected with plasmids of pCMV or pCMV-CD36 overnight using Hieff Trans™ Reagent (Yeasen Biotechnology Co., Ltd., shanghai, China), followed by further treatment.
CD36-knockdown vector was constructed using CRISPR-CasRx methods. Simply, the sequence of gRNA was designed as follows: forward, 5′-AAACACACAGGGATTCCTTTCA GATT-3′ and backward, 5′-AAAAAATCTGAAAGGAATCCCTGTGT-3′. CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase. The obtained CasRx-CD36 plasmid was verified with U6 sequencing primer (Konermann et al. 2018 ). For knockdown experiment, cells at ~ 60% confluence were transfected with EF1a-CasRx-2A-EGFP (pXR001, #109049, Addgene) and CasRx gRNA cloning backbone or CasRx-CD36 plasmid for 12 h using Hieff Trans™ Liposomal Transfection Reagent and then received the indicated treatment.

Lentiviral vectors for the overexpression of circ_0021350 (pLVX-circ_0021350), PIK3R3 (pLVX-PIK3R3), and shRNAs of circ_0021350 were synthesized by Miaolingbio (Wuhan, China). Lipofectamine 3000 (Invitrogen, USA) was used for plasmid transfection according to the manufacturer’s instructions. The cells transfected with the lentiviral vector were screened with 2.0 µg/mL puromycin for six weeks. Circ_0021350 siRNAs, siRNA negative control (siRNA-NC), miR-1207-3p mimics, inhibitors, mimic negative control (mimic-NC), and inhibitor negative control (inhibitor-NC) were synthesized by Biomics Biotechnologies (Beijing, China) and transfected using HieffTrans reagent (Yeasen Biotechnology, China).