Sihur

CELF1 or HuR was silenced by transfection with a specific siRNA as described previously (Zou et al., 2010 (link); Xiao et al., 2011 (link)). The siRNAs specifically targeting mRNAs encoding CELF1 (siCUGBP1) or HuR (siHuR) and control-siRNA (C-siRNA) were purchased from Santa Cruz. For each 60-mm cell culture dish, 15 μl of the 20 μM stock duplex siCELF1, siHuR, or C-siRNA was used. Forty-eight hours after transfection using Lipofectamine, cells were harvested for analysis.

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.