FIGURE 2:

CUGBP1 silencing enhances occludin translation and promotes the epithelial barrier function. (A) Representative immunoblots of CUGBP1 and occludin proteins. After cells were transfected with either siRNA targeting the CUGBP1 mRNA CR (siCUGBP1) or C-siRNA for different times, whole-cell lysates were harvested for Western blot analysis. (B) Levels of occludin mRNA as measured by Q-PCR analysis in cells described in panel (A). The data were normalized to GAPDH mRNA levels and shown as the means ± SEM of data from triplicate experiments. (C) Newly translated occludin protein in cells transfected with siCUGBP1 or C-siRNA for 48 h as measured by l-[³⁵S]methionine and l-[³⁵S]cysteine/IP assays. Top, representative immunoblots of newly synthesized occludin; bottom, quantitative analysis of the immunoblotting signals as measured by densitometry. Values are means ± SEM of data from three separate experiments. * p < 0.05 compared with cells transfected with C-siRNA. (D) Changes in occludin translation efficiency as measured by using pGL3-Luc-Occl-3′-UTR reporter assays in cells described in panel (C). Twenty-four hours after cells were transfected with the Luc-Occl-3′-UTR or pGL3-Luc, the levels of luciferase activity were examined and normalized to the mRNA levels to calculate the translation efficiencies. Values were expressed as means ± SEM of data from three separate experiments; * p < 0.05 compared with cells transfected with C-siRNA. (E) Changes in TEER (left panel) and FITC–dextran paracellular permeability (right panel) in cells transfected with siCUGBP1 alone or cotransfected with siCUGBP1 and siRNA targeting occludin (siOccludin). TEER assays were performed on 12-mm Transwell filters 48 h after transfection as described in Materials and Methods; paracellular permeability was assayed by using the membrane-impermeable trace molecule FITC–dextran that was added to the insert medium. Values are the means ± SEM of data from six samples. *,+ p < 0.05 compared with cells transfected with C-siRNA or siCUGBP1, respectively. (F) Changes in TEER and FITC–dextran paracellular permeability in cells transfected with the CUGBP1 expression vector or cotransfected with CUGBP1 and occludin expression vectors. *, # p < 0.05 compared with cells transfected with control vector (vector) or CUGBP1 expression vector alone, respectively. (G) Changes in the levels of CUGBP1 and occludin proteins in cells described in (E) and (F).