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.
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.