Figure 3.
Figure 3. Widespread EndMT in Erk1−/− Erk2iEC−/− mice. (a) TGFβ staining in the kidney, heart, and liver of Erk1−/− Erk2iEC−/− mice 3 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrowheads point to endothelial cells expressing TGFβ. The small picture represents the single channel of the large picture. (b) pSmad2 staining in kidney, heart, and liver from Erk1−/− Erk2iEC−/− mice 4 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrows point to endothelial cells with no or low pSmad2 staining. Arrowheads point to endothelial cells with intense pSmad2 staining. Small pictures are a higher magnification of the field in the white square in the large pictures. (c) Collagen-I staining in kidney, heart, and liver from Erk1−/− Erk2iEC−/− mice 3 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrowheads point to endothelial cells expressing collagen-I. The small picture represents the single channel of the large picture. (d) GFP and sm22α staining in the kidney, heart, and liver of Cdh5Cre-mTmG mice (denoted WT) and Erk1−/− Erk2iEC−/− mTmG mice 4 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrowheads point to endothelial cells expressing sm22α. The small picture represents the single channel of the large picture. (e) Quantification of the proportion of VE-cadherin+ cells expressing sm22α by FACS (n = 4 mice per genotype and per time point). *, P < 0.05; **, P < 0.01; Kruskal–Wallis test followed by Dunn’s multiple comparisons to WT mice. (f) Evaluation of Tgfb1, Tgfb2, and Tgfb3 mRNA expression by qPCR in HUVECs after Erk1 and Erk2 knockdown (n = 6). ***, P < 0.001; Mann–Whitney U test. (g) Active and latent TGFβ2 concentration in conditioned media from HUVECs knocked down for ERK1 and ERK2 (n = 9). ***, P < 0.001; Mann–Whitney U test. (h) Expressions of endothelial markers (VEGFR2 and NOS3) and mesenchymal markers (N-cadherin, collagen-I, and sm22α) by Western blot in HUVECs knocked down for ERK1 and ERK2 and HUVECs treated with TGF-β2 at 10 ng/ml for 4 d (representative blot from n = 3). Error bars represent SEM.

Widespread EndMT in Erk1−/− Erk2iEC−/− mice. (a) TGFβ staining in the kidney, heart, and liver of Erk1−/− Erk2iEC−/− mice 3 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrowheads point to endothelial cells expressing TGFβ. The small picture represents the single channel of the large picture. (b) pSmad2 staining in kidney, heart, and liver from Erk1−/− Erk2iEC−/− mice 4 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrows point to endothelial cells with no or low pSmad2 staining. Arrowheads point to endothelial cells with intense pSmad2 staining. Small pictures are a higher magnification of the field in the white square in the large pictures. (c) Collagen-I staining in kidney, heart, and liver from Erk1−/− Erk2iEC−/− mice 3 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrowheads point to endothelial cells expressing collagen-I. The small picture represents the single channel of the large picture. (d) GFP and sm22α staining in the kidney, heart, and liver of Cdh5Cre-mTmG mice (denoted WT) and Erk1−/− Erk2iEC−/− mTmG mice 4 wk after tamoxifen injections (representative of n = 4 mice per genotype). Scale bars, 50 µm. Arrowheads point to endothelial cells expressing sm22α. The small picture represents the single channel of the large picture. (e) Quantification of the proportion of VE-cadherin+ cells expressing sm22α by FACS (n = 4 mice per genotype and per time point). *, P < 0.05; **, P < 0.01; Kruskal–Wallis test followed by Dunn’s multiple comparisons to WT mice. (f) Evaluation of Tgfb1, Tgfb2, and Tgfb3 mRNA expression by qPCR in HUVECs after Erk1 and Erk2 knockdown (n = 6). ***, P < 0.001; Mann–Whitney U test. (g) Active and latent TGFβ2 concentration in conditioned media from HUVECs knocked down for ERK1 and ERK2 (n = 9). ***, P < 0.001; Mann–Whitney U test. (h) Expressions of endothelial markers (VEGFR2 and NOS3) and mesenchymal markers (N-cadherin, collagen-I, and sm22α) by Western blot in HUVECs knocked down for ERK1 and ERK2 and HUVECs treated with TGF-β2 at 10 ng/ml for 4 d (representative blot from n = 3). Error bars represent SEM.

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