Dh5α cells

A 2.369 kb fragment containing a 5ʹ VEGFA sequence from −2304 to +65 relative to the transcription initiation site was amplified by PCR using Q5 High-Fidelity DNA Polymerase (NEB, United States). The forward primer with a SalI site was 5ʹ-GATGTCGACTTGCTGGGTACCACCATGGA-3ʹ, and the reverse primer, which had a XbaI site, was 5ʹ-GATTCTAGACAGAGCGCTGGTGCTAGCC-3ʹ. After digestion by QuickCut restriction endonucleases (Takara, United States), a DNA Ligation Kit (Takara, United States) was used to insert the PCR sequences into the SalI and XbaI sites of pEZX-FR01 (GeneCopoeia, United States), which contains a Rinella luciferase (Rluc) coding sequence with a CMV promoter and a promoter-less firefly luciferase (HLuc) coding sequence.
These recombinant plasmids were transfected into DH5α cells (TSINGKE, China) and amplified in nutrition agar plate (Solarbio, China) culture with kanamycin monosulfate (50 μg·mL⁻¹, Solarbio, China) to select different monoclonal bacterial colonies. Then, the selected single clones were cultured in LB medium (Solarbio, China) with kanamycin. The recombinant plasmids were purified by a plasmid extraction kit (TIANGEN, China). All plasmid constructs were verified by direct sequencing (Sangon Biotech, Chengdu). The reporter plasmid was designated pEZX-VEGFA.

The pCDH-EF1-copGFP plasmid and pHAGE-puro plasmid were purchased from GenePharma (Shanghai, China). The human IL-15 overexpression plasmid was purchased from Genomeditech (Shanghai, China). For construction of the mouse IL-15 overexpression vectors, targeted primers were designed to amplify the open reading frame (ORF) of mouse IL-15 using 2 × Phanta Max Master Mix (Cat: P515-01; Vazyme, Nanjing, China) (Table S2). Mlu-I and Bmt-I (Thermo Fisher Scientific, USA) were used to digest the pHAGE-puro plasmid. The mouse IL-15-Flag (ORF) sequence was ligated into the resultant plasmid. Primers targeting human IL-15 were designed to construct the short hairpin RNA (shRNA)-IL-15 plasmid (Table S2). The pCDH-EF1-copGFP plasmid was digested with Age I and EcoR I (Thermo Fisher Scientific, USA). The constructed plasmid was subsequently transformed into DH5α cells (Tsingke, Wuhan, China), after which the resulting constructs were sequenced.