Figure 4. Decreased expression of β-catenin in FoxM1-deficient ECs. (A) Quantitative RT-PCR analysis of expression of components of endothelial AJs. At 65 h after transfection of either FoxM1 siRNA or scRNA, confluent HMVEC-L were lysed for RNA isolation. mRNA levels of the indicated genes were quantified by quantitative RT-PCR analysis. Data are expressed as mean ± SD (error bars; n = 3). *, P < 0.05 versus scRNA. The experiment was performed three times with similar results. (B and C) Western blot analysis demonstrating decreased protein levels of β-catenin in FoxM1-deficient HMVEC-L. At 65 h after transfection with either human FoxM1 siRNA (siRNA) or scRNA, confluent HMVEC-L were lysed for Western blot analysis of each protein. Anti-actin was used as a loading control (B). The experiment was performed three times with similar results. β-catenin expression was quantified by densitometry analysis (C). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. (D and E) Subcellular localization of endothelial AJ proteins at baseline and after thrombin challenge. At 65 h after transfection, confluent HMVEC-L monolayers were collected for cell fractionation. Each membrane (M) or cytosolic (C) fraction (7.5 µg per lane) was loaded for detection of each protein (D). The experiment was performed three times with similar results. β-catenin localization in the membrane fraction versus cytosolic fraction was quantified by densitometry (E). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.01 versus membrane expression at time 0 (basal); #, P < 0.05 versus cytosolic expression at time 0; ƒ, P < 0.05 versus scRNA-membrane expression at time 0.
Figure 4.

Decreased expression of β-catenin in FoxM1-deficient ECs. (A) Quantitative RT-PCR analysis of expression of components of endothelial AJs. At 65 h after transfection of either FoxM1 siRNA or scRNA, confluent HMVEC-L were lysed for RNA isolation. mRNA levels of the indicated genes were quantified by quantitative RT-PCR analysis. Data are expressed as mean ± SD (error bars; n = 3). *, P < 0.05 versus scRNA. The experiment was performed three times with similar results. (B and C) Western blot analysis demonstrating decreased protein levels of β-catenin in FoxM1-deficient HMVEC-L. At 65 h after transfection with either human FoxM1 siRNA (siRNA) or scRNA, confluent HMVEC-L were lysed for Western blot analysis of each protein. Anti-actin was used as a loading control (B). The experiment was performed three times with similar results. β-catenin expression was quantified by densitometry analysis (C). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. (D and E) Subcellular localization of endothelial AJ proteins at baseline and after thrombin challenge. At 65 h after transfection, confluent HMVEC-L monolayers were collected for cell fractionation. Each membrane (M) or cytosolic (C) fraction (7.5 µg per lane) was loaded for detection of each protein (D). The experiment was performed three times with similar results. β-catenin localization in the membrane fraction versus cytosolic fraction was quantified by densitometry (E). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.01 versus membrane expression at time 0 (basal); #, P < 0.05 versus cytosolic expression at time 0; ƒ, P < 0.05 versus scRNA-membrane expression at time 0.

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