Electron microscopy pictures show an activated endothelial phenotype in tissues from BAMBI^−/− as compared to BAMBI^+/+ mice.

<p><b>A</b>. Myocardium of the BAMBI<b>⁻</b>^/<b>−</b> mice is notable for the number of endothelial cells per capillary cross-section and the prominent nuclei. In BAMBI^+/+ mice myocardial capillaries show one endothelial cell per cross-section, while those from BAMBI<b>⁻</b>^/<b>−</b> mice show two nuclei per cross-section in 70% of the capillaries (scale bar 2 µm). <b>B</b> and <b>C</b>. glomeruli from BAMBI<b>⁻</b>^/<b>−</b> mice show a prominent number of endothelial cells, which also appear swollen as compared to those from BAMBI^+/+ mice. The glomerular capillary lumen is almost obliterated by the endothelial cells in the BAMBI<b>⁻</b>^/<b>−</b> mice <i>vs.</i> BAMBI^+/+ (low magnification).The activated endothelial phenotype in the BAMBI<b>⁻</b>^/<b>−</b> glomeruli is also apparent at higher magnification (bottom row). <b>D.</b> Peritubular capillaries of BAMBI<b>⁻</b>^/<b>−</b> mice also show a prominent and swollen endothelial cells impinging on the capillary lumen as compared to BAMBI ^+/+ mice.</p

Albumin creatinine ratio, and BUN.

<p>Urinary albumin to creatinine ratios from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice at baseline and two weeks after unilateral nephrectomy. BUN levels from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice at baseline and 2 weeks after unilateral nephrectomy. Values are means ± SEM for the number of animals indicated in brackets.</p

Kidney weights per body weight and glomerular and endothelial areas in the unilateral nephrectomy specimens (day 0) and in the remaining kidneys after 14 days of compensatory hypertrophy (day 14).

<p><b>A.</b> Kidney weights per body weight are comparable between BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice and increase to a comparable degree during the 14 days of compensatory hypertrophy <b>B</b> and <b>C</b>. Glomerular areas <b>B.</b> and endothelial (isolectin B4 positive) areas <b>C</b>. are larger in BAMBI<b>⁻</b>^/<b>−</b> than in BAMBI^+/+ mice in the unilateral nephrectomy specimens and the differences become even bigger after 14 days of compensatory renal hypertrophy. Data are mean ± SEM, <i>n</i> = 5 mice per group.</p

<i>In vivo</i> angiogenesis by modified-matrigel implantation assay is enhanced in BAMBI^−/− mice in comparison to their BAMBI^+/+ littermates.

<p><b>A. </b><i>In vivo</i> angiogenesis was evaluated under basal conditions and in the presence of VEGF and FGF in the implants in BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice. In-growth of endothelial cells was evaluated after 12 days by isolectin B4 fluorescence determination. Results are means ± SEM from 4 animals per group. # <i>P</i><0.05 <i>vs</i>. respective basal values; * <i>P</i><0.05 BAMBI<b>⁻</b>^/<b>−</b><i>vs</i>. BAMBI^+/+. <b>B.</b> Comparable experiments except that TGFβ ± LY364947 or LY364947 alone were added to the matrigel implants. Results are means ± SEM from 5 mice per group. * <i>P</i><0.05 comparing respective basal <i>vs.</i> growth factor stimulated values and # <i>P</i><0.05 for group comparison of BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice in the same condition.</p

Expression of BAMBI in different mouse organs.

<p><b>A.</b> Levels of mRNA (relative copy number) were determined in different organs from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice. Two different primer pairs were used, spanning either exons 2–3 (top panel) or exons 1–2 (bottom panel) in order to exclude transcripts from a potential second start site in the BAMBI gene. In the tissues from the BAMBI<b>⁻</b>^/<b>−</b> mice, and using either primer pair, the levels of mRNA for BAMBI were indistinguishable from the RT minus or blank controls. Results are means ± SEM from triplicate determinations performed in two sets of mice. <b>B.</b> Immunofluorescence staining for BAMBI performed on different organs from BAMBI^+/+ and BAMBI<b>⁻</b>^/<b>−</b> mice. Organs are by rows: A: lung; B: heart; C: liver; D: kidney (g indicates glomeruli); E: femoral artery and vein; F: veins with accompagnying lymphatic vessel (LYVE-1 staining). Magnification 400x for A, B, C and F. 200x for D and E. BAMBI staining in red (first column A–F BAMBI^+/+ mouse tissues), second column endothelial markers in green (MECA32, BS 4 Isolectin ) and the merge in the third column in yellow. BAMBI co-localizes with endothelial markers in all tissues, but is negative in tissues from BAMBI<b>⁻</b>^/<b>−</b> mice (Inserts first column row A–D). Lymphatic endothelium stains with Lyve-1 antibody, but not with BAMBI antibody or MECA 32 (bottom row).</p

BAMBI influence alternative TGFβ signaling.

<p><b>A.</b> and <b>D.</b> mRNA levels for BAMBI, ALK1,TβRI and TβRII in HUVEC. <b>A.</b> HUVEC cell line transfected with lentiviral system containing either the empty vector or the BAMBI over-expressing vector; <b>D.</b> primary HUVEC without or with transfection of scrambled RNA or siRNA for BAMBI knock-down). Results are means ± SEM from three experiments each and are expressed as copy number per 18S control. <b>B</b> and <b>E.</b> Analysis by Wesern blots of the phosphorylation of SMAD1/5, SMAD3 and ERK1/2 in HUVEC with either BAMBI over-expression or knock-down. <b>B.</b> BAMBI over-expression transfected or empty vector transfected HUVEC were incubated with TGFβ ± LY364947 or RDEA-119 for 15 min before extraction of the proteins and analysis by Western blot as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039406#s2" target="_blank">Methods</a>. Each blot was analyzed with antibodies specific for the phosphorylated and the total form of proteins and GAPDH, to calculate the ratio of phospho- to total protein. A representative blot is shown. The experiments with LY364947 and RDEA-119 were carried out in parallel and run on separate blots. For convenience of presentation, the lanes from the blots containing the TGFβ plus RDEA-119 samples were cut out and added to the respective blots from the parallel experiment with LY364947. <b>E</b>. Comparable experiments as in (<b>B</b>), except that HUVEC had been transfected with either scrambled siRNA or siRNA for BAMBI prior to the experimental incubations. <b>C</b> and <b>F.</b> The bar graphs show the results of the activation ratios for the different signaling molecules calculated from the densitometry readings. Each bar represents the mean ± SEM of 6 separate series of experiments carried out at basal and TGFβ stimulated conditions, in 4 series LY364947 was tested in addition, and in 3 series RDEA-119. # refers to <i>P</i><0.05 by group comparison, and * to <i>P</i><0.05 by comparison to the respective basal values.</p