Prl tk renilla luc plasmid

THP-1 cells from a human monocytic leukemia cell line were maintained in RPMI-1640 medium (Sigma), containing 10% FCS, 100 IU/ml penicillin G, and 100 μg/ml streptomycin, at 37 °C under a 5% CO₂ atmosphere. Transfection experiments were performed using GenomONE-GX (Ishihara Sangyo). THP-1 cells were co-transfected with the human ENPP1 expression plasmid, the pGL4-IFN-β-promoter-Luc plasmid, and the pRL-TK Renilla-Luc plasmid (Promega). At 24 h after transfection, the cells were lysed and the luciferase activity was measured with a GloMax 20/20 luminometer (Promega), using the Dual-Luciferase Reporter Assay System (Promega). The cell lysates were then immunoblotted with an anti-ENPP1 antibody (Cell Signaling, Cat. No. 2061, 1:1000 dilution) or an anti-tubulin antibody (Cedarlane, Cat. No.CLT9002, 1:3000 dilution). The uncropped images are shown in Supplementary Figure 3. All cell lines were negative for mycoplasma contamination.

HEK293T cells were cultured in 96-well plates and transfected with 1 ng empty vector, 0.1 ng pCAGGS-STING-AGIA, or 1 ng pCAGGS-STING-S4D-AGIA together with 4 ng pGL4-ISRE-promoter-Luc plasmid (Promega) and 1 ng pRL-TK-Renilla-Luc plasmid (Promega). After the cells were transfected for 6 h, they were pretreated with DMSO (0.1%) or pomalidomide in culture medium for 16 h. The cells were stimulated with 2 µg/ml cGAMP in permeabilization buffer [50 mM HEPES (pH 7.2–7.5), 100 mM KCl, 3 mM MgCl2, 0.1 mM dithiothreitol (DTT), 85 mM sucrose, 1 mM ATP, 0.2% BSA, 10 µg/ml digitonin) for 3 h, and then lysed with 45 µl 1× Passive Lysis Buffer, and the Firefly and Renilla luciferase activity was measured on a Glomax luminometer using the dual-luciferase reporter assay system (Promega).