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. 2016 Nov 14;12(4):194–216. doi: 10.1080/15476278.2016.1252887

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© 2016 Taylor & Francis

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FIGURE 3. — Rac GTPase Inactivation Results in Disruption of the Basement Membrane and Cellular Polarity. (A) ICC for collagen IV (green) and DAPI staining (blue) following 96-hour culture of E13 SMGs treated with vehicle or 10 µM EHT indicate Rac1 inactivation reduces collagen IV in the basement membrane surrounding epithelial buds. Scale bars, 10 µm. (B and C) Representative western analysis and quantification performed following 96-hour culture with or without 10 µM EHT demonstrates a slight loss in collagen IV levels (42% reduction) in E13 SMG with EHT treatment (p = 0.66, n ≥ 3 Experiments). (D) ICC following 96-hour culture with or without 10 µM EHT for Par-1b (cyan) and collagen IV (green) with DAPI staining (blue) demonstrates a loss in Par-1b levels in the outer epithelial cells with EHT treatment. Scale bars, 20 µm. (E and F). Western analysis for Par-1b following EHT treatment indicates a loss of total Par-1b within the gland (25% reduction), quantified relative to GAPDH (n ≥ 3). (G) SMGs were treated with either Par-1b or NT control siRNA, and Western analysis was performed to detect collagen IV and GAPDH. (H) Quantification of collagen IV levels demonstrated 60% reduction in Par-1b levels relative to GAPDH (n = 3). (I) E13 epithelial rudiments treated with vehicle or Par-1b WT adenovirus were recombined with untreated mesenchyme, and cultured for 96 hours either with or without 7.5 µM EHT. ICC was performed for collagen IV (green) and Par-1b (cyan), together with DAPI staining for nuclei (blue). Par-1b WT adenovirus treatment shows increased collagen IV levels relative to control, and glands treated with both WT Par-1b adenovirus and EHT show an intermediate phenotype indicating partial rescue of collagen IV with exogenous Par-1b in the presence of EHT. Scale bars, 10 µm.

graphic file with name kogg-12-04-1252887-g006.jpg — Rac GTPase Inactivation Results in Disruption of the Basement Membrane and Cellular Polarity. (A) ICC for collagen IV (green) and DAPI staining (blue) following 96-hour culture of E13 SMGs treated with vehicle or 10 µM EHT indicate Rac1 inactivation reduces collagen IV in the basement membrane surrounding epithelial buds. Scale bars, 10 µm. (B and C) Representative western analysis and quantification performed following 96-hour culture with or without 10 µM EHT demonstrates a slight loss in collagen IV levels (42% reduction) in E13 SMG with EHT treatment (p = 0.66, n ≥ 3 Experiments). (D) ICC following 96-hour culture with or without 10 µM EHT for Par-1b (cyan) and collagen IV (green) with DAPI staining (blue) demonstrates a loss in Par-1b levels in the outer epithelial cells with EHT treatment. Scale bars, 20 µm. (E and F). Western analysis for Par-1b following EHT treatment indicates a loss of total Par-1b within the gland (25% reduction), quantified relative to GAPDH (n ≥ 3). (G) SMGs were treated with either Par-1b or NT control siRNA, and Western analysis was performed to detect collagen IV and GAPDH. (H) Quantification of collagen IV levels demonstrated 60% reduction in Par-1b levels relative to GAPDH (n = 3). (I) E13 epithelial rudiments treated with vehicle or Par-1b WT adenovirus were recombined with untreated mesenchyme, and cultured for 96 hours either with or without 7.5 µM EHT. ICC was performed for collagen IV (green) and Par-1b (cyan), together with DAPI staining for nuclei (blue). Par-1b WT adenovirus treatment shows increased collagen IV levels relative to control, and glands treated with both WT Par-1b adenovirus and EHT show an intermediate phenotype indicating partial rescue of collagen IV with exogenous Par-1b in the presence of EHT. Scale bars, 10 µm.