β galactosidase enzyme assay system

pDMVEC were transfected using pcDNA3.1-FLAG-NF-κB p65 (pc-p65) or control vectors as described previously [48] (link). Cells were also transfected in 12-well plates using Lipofectamine 2000 (Invitrogen) for 24 to 48 h and constructs for overexpression of miR-K12-1 and miR-K12-11 as previously described [49] (link). For RNA interference, pDMVEC were transfected for 48 h with either SK2-, ORF50-, or control (non-target)-siRNAs using ON-TARGET plus SMART pool (Dharmacon) and DharmaFECT Transfection Reagent (Dharmacon) according to the manufacturer’s instructions. Transfection efficiency was normalized through co-transfection of a lacZ reporter construct and determination of β-galactosidase activity using a commercial β-galactosidase enzyme assay system according to the manufacturer’s instructions (Promega). For some experiments, transfection efficiency was further assessed on a single-cell level through co-transfection with a green fluorescent protein (GFP) construct (pEGFP-N1) and subsequent flow cytometry analysis as described previously [50] (link). This procedure was performed using pDMVEC and HEK293A cells (as a positive control), since the transfection efficiency of GFP for HEK293A cells approaches 90%. Using this method, we confirmed the pDMVEC transfection efficiency of approximately 50% of cells in our experiments (Fig. S2).

ISG15 and ISG20 ON-TARGET plus SMART pool siRNA, or negative control siRNA (n-siRNA) (Dharmacon), were delivered using the DharmaFECT transfection reagent according to the manufacturer's instructions. For plasmid transfection, PDLF were transfected in 12-well plates with miR-K12-1 recombinant construct or control vector as previously described [28 (link)] by using Lipofectamine 3000 (Invitrogen) for 48 h. Transfection efficiency was normalized through co-transfection of a lacZ reporter construct and determination of β-galactosidase activity using a commercial β-galactosidase enzyme assay system according to the manufacturer's instructions (Promega).

The 315 bp wild‐type 3′‐UTR of human RUNX3 mRNA containing miR‐532‐5p binding site was amplified by PCR and inserted into the SpeI/HindIII sites of pMIR‐REPORT^™ luciferase reporter plasmid to generate pMIR‐RUNX3/wt plasmid. The complementary sequence for miR‐532‐5p seed sequence in RUNX3 3′‐UTR was mutated using QuickChange site‐directed mutagenesis kit with the pMIR‐RUNX3/wt plasmid as template. The mutant was named as pMIR‐RUNX3/mut plasmid. The cells were cotransfected with miR‐532‐5p mimics and pMIR‐RUNX3/wt or pMIR‐RUNX3/mut transiently. Meanwhile, pMIR‐REPORT^™ β‐gal control plasmid was cotransfected to normalize variability because of differences in cell viability and transfection efficiency. 48 hrs later, luciferase activity and β‐galactosidase activity were determined using a dual luciferase reporter assay system and β‐Galactosidase enzyme assay system (both from Promega, Madison, WI, USA).

pGL3-Beclin-1(−644/+197, −277/+197, −58/+197)-luciferase reporter plasmids were kindly provided by Dr. Mujun Zhao (Institute of Biochemistry and Cell Biology, Shanghai, China). pRKbetaGAL containing the CMV promoter-driven β-galactosidase gene was used to normalize the transfection efficiency. A pGL3-Beclin-1-luciferase reporter plasmid and pRKbetaGAL were co-transfected into cultured Vero cells. After 24 hr of incubation, ARV S1133 at an MOI of 5 was added and the mixture incubated for an additional 24 hr. The luciferase activities were quantified using a Luciferase assay system (Promega), and the β-galactosidase activities were quantified using a β-galactosidase Enzyme Assay System (Promega). Luciferase activity was normalized to β-galactosidase activity to calculate the transfection efficiency [42 (link)].