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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 1. — Decreased Branching Morphogenesis of the Mouse Submandibular Salivary Gland with Pharmacological Inactivation of Rac GTPase activity or siRNA Knockdown of Rac1. Representative brightfield images of E13 SMG organ explants cultured for 96 hours (A) with vehicle control or 10 µM EHT, (B) non-targeting (NT) siRNA or Rac-1 siRNA indicate Rac1 expression and activity are both required for proper branching morphogenesis of the developing SMG. Scale bars, 100 µm. (C) Morphometric analysis of control and 10 µM EHT-treated E13 SMGs show a statistically significant decrease in the number of buds in EHT-treated glands (**p ≤ 0 .01) and (D) morphometric analysis of Rac1 siRNA-treated glands show a decreasing trend in bud number relative to NT siRNA-treated glands. (E) Representative western blots and (F) quantification of Rac1 levels in glands treated with 10 µM EHT or Rac1 siRNA, normalized to GAPDH, and compared to controls shows a decrease after siRNA treatment (46% reduction) but not with EHT treatment, as expected (n ≥ 3 experiments) (n.s.). (G and H) Immunocytochemistry (ICC) for Rac1 (cyan) and ECAD (green), with DAPI (blue) staining for nuclei shows Rac1 localized primarily in the outer epithelial cells with no change in localization following 10 µM EHT treatment for 96 hours but a decrease in Rac1 levels within the ECAD⁺ epithelium after Rac1 siRNA treatment. Scale bars, 10 µm.

graphic file with name kogg-12-04-1252887-g002.jpg — Decreased Branching Morphogenesis of the Mouse Submandibular Salivary Gland with Pharmacological Inactivation of Rac GTPase activity or siRNA Knockdown of Rac1. Representative brightfield images of E13 SMG organ explants cultured for 96 hours (A) with vehicle control or 10 µM EHT, (B) non-targeting (NT) siRNA or Rac-1 siRNA indicate Rac1 expression and activity are both required for proper branching morphogenesis of the developing SMG. Scale bars, 100 µm. (C) Morphometric analysis of control and 10 µM EHT-treated E13 SMGs show a statistically significant decrease in the number of buds in EHT-treated glands (**p ≤ 0 .01) and (D) morphometric analysis of Rac1 siRNA-treated glands show a decreasing trend in bud number relative to NT siRNA-treated glands. (E) Representative western blots and (F) quantification of Rac1 levels in glands treated with 10 µM EHT or Rac1 siRNA, normalized to GAPDH, and compared to controls shows a decrease after siRNA treatment (46% reduction) but not with EHT treatment, as expected (n ≥ 3 experiments) (n.s.). (G and H) Immunocytochemistry (ICC) for Rac1 (cyan) and ECAD (green), with DAPI (blue) staining for nuclei shows Rac1 localized primarily in the outer epithelial cells with no change in localization following 10 µM EHT treatment for 96 hours but a decrease in Rac1 levels within the ECAD⁺ epithelium after Rac1 siRNA treatment. Scale bars, 10 µm.