Mid1 silencer select sirna

eEnd.2 cells (2 × 10⁵/well) were seeded into 6-well plates for 24 h and transfected when the confluence was about 70% with silencer select negative control siRNA or Mid1 silencer select siRNA (Ambion, CA, USA) by use of TransIT-TKO transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. Then, 24 h after transfection, transfected cells were stimulated with 100 ng/mL murine TNF-α (PeproTech) for 1 h or 3 h, and then cells were collected for subsequent analysis. HLMVEC transfections were performed using human Mid1 silencer select siRNA (Ambion, CA, USA) according to the manufacturer’s instructions, and stimulation was performed with 10 ng/mL recombinant human TNF-α (Gibco).

For detection of ICAM-1 expression, eEnd.2 cells were transfected with silencer select negative control siRNA or Mid1 silencer select siRNA (Ambion), as described above, and then stimulated with 100 ng/mL TNF-α (PeproTech) for 3 h. Cells were washed twice with PBS, and single cell suspension was prepared by digestion with Accutase (A6964, Sigma-Aldrich). Samples were centrifuged (400× g, 5 min) and pellets were resuspended and fixed with 2% formaldehyde. After washing two times with PBS containing 2% FBS, cells were incubated with an anti-CD16/CD32 for 10 min to block FcϒIII/IIRs and then incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-mouse ICAM-1 (YN1/1.7.4; Biolegend, London, UK) antibody for 20 min. Mean fluorescence intensity (MFI) of ICAM-1 expression was detected using Cytoflex flow cytometer (Becton Dickinson, Mountain View, CA, USA).