Pcdna3.1 p2a egfp vector

The coding regions of CP204L (p30) and E183L (p54) from ASFV isolate Pig/HLJ/2018 (Genbank accession No. MK333180.1) were codon-optimized according to human codon usage, synthesized by Genscript (Nanjing, China), and cloned into pcDNA3.1(+)-P2A-eGFP vector, respectively. These two plasmids were digested with restriction enzymes EcoR V and Xho I, and the codon-optimized p30 and p54 genes were gel-purified and cloned into the pCAGGS-GLuc-FLAG vector using T4 DNA ligase (Vazyme Biochem, Nanjing, China). Finally, the plasmids for the expression of the fusion proteins, GLuc-p30 and GLuc-p54, were designated as pCAGGS-GLuc-p30 and pCAGGS-GLuc-p54 (Figure 2A). The correct insertion of p30 and p54 coding sequences was verified via DNA sequencing by Genscript (Nanjing, China).

The FGFR2-AHCYL2, FGFR2-SH3GLB1, and FGFR2-BICC1 fusion genes were generated and cloned into the pcDNA3.1(+)P2A-eGFP vector by GenScript (New Jersey, U.S). GenScript used site-directed mutagenesis to introduce the p.V564F and p.E565A SNVs into the FGFR2-AHCYL2 fusion. NIH3T3 cells were stably transfected with 2 µg of the linearized vectors using Effectene® transfection reagent (Qiagen, Hilden, Germany). Single clones were selected using Geneticin (G418 disulfate salt; Biochrom, Berlin, Germany). Empty pcDNA3.1(+)P2A-eGFP transfected NIH3T3 were used as control.

To overexpress PD-L1-lnc, the full-length cDNA of PD-L1-lnc was synthesized and cloned into pcDNA3.1-P2A-eGFP vector (GenScript, China). To suppress PD-L1-lnc, the PD-L1-lnc shRNA vectors were synthesized and then cloned into pLKO.1 vector (GenScript). The siRNA target sequences were listed in Table S3. Cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction.