C sirna

Lenti-SPRY4-IT1 was custom made by AMSBIO, in which SPRY4-IT1/GFP expression was under the control of the suCMV-promoter. Lenti-SPRY4-IT1 and C-lentiviral were packaged in lentiviral production cells, concentrated by ultracentrifugation, resuspended in phosphate-buffered saline (PBS), and used to increase SPRY4-IT1 in vivo as described (Scherr et al., 2007 (link); Feng et al., 2012 (link)). An expression vector containing SPRY4-IT1 cDNA under control of pCMV-promoter was constructed (Liu et al., 2009 (link)) and used to increase SPRY4-IT1 in Caco-2 cells; claudin-1 and occludin expression vectors were obtained from Origene (Rockville, MD).
Expression of SPRY4-IT1 and HuR was silenced by transfection with specific siRNA as described (Cui et al., 2011 (link); Liu et al., 2015 (link)). The siSPRY4-IT1, siHuR, and C-siRNA were purchased from Santa Cruz Biotechnology. For each 60-mm cell culture dish, 15 μl of the 20 μM stock duplex siSPRY4-IT1, siHuR, or C-siRNA was used. Forty-eight hours after transfection using Lipofectamine, cells were harvested for analysis.

MMC were transfected with 60 pmol SOCS1 or control siRNA (csiRNA) (Santa Cruz) using 6 µL of transfection reagent for 6 hours after which RPMI medium conatining20% FBS was added. After 18 hours, media was replaced with RPMI/10% FBS and incubated for 24 hours before stimulation with mouse IFN-γ as described previously. SOCS1 protein expression was decreased by 66%±12% with siRNA treatment; SOCS1 protein expression is described as % inhibition or restoration based on adjuvant control.

Control small interfering RNA (C.siRNA; Cat# SC‐37007) and siRNA targeted against Peli1 (Peli1.siRNA; Cat# SC‐44401) were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX). The HUVECs were treated with either C.siRNA or Peli1.siRNA (60 nmol/L) for 48 hours and then treated with VEGF. Matrigel with reduced serum (Cat# 354230, Corning Incorporated, Corning, NY) was dissolved at 4°C overnight, after which a μ‐Slide plate (ibidi USA, Inc, Madison, WI) was prepared with 10 μL Matrigel in each well and incubated at 37°C for 30 minutes. After incubation of the plates, 50 μL of treated cells (1.5×10⁵ cells/mL) were seeded onto the gel. Tube formation was assessed after 8 hours of seeding using an Olympus QColor 3TM digital camera mounted on an Olympus BH2 microscope. Each well was photographed at ×200 magnification.11