(XLSX) Click here for additional data file.(583K, xlsx) S4 DataData underlying Fig 4A and 4G, S5D and S9 Figs. (predicted molecular weight BMPR2wt approximately 140C150 kDa; BMPR2approximately 130 kDa) (left). Data 4-Aminobenzoic acid are presented as mean + SD relative to lane 1 (one-way ANOVA with post hoc Bonferroni, = 4 independent experiments). (F) Cell surface biotinylation at primary amines followed by precipitation using Streptavidin in indicated clones (upper) or Cos7 cells overexpressing indicated BMPR2 constructs (lower). (G) Confocal microscopy of cells transiently transfected with a myc-tagged BMPR2E2 construct. Cells were immunostained with anti-BMPR2 antibody (green) and anti-myc antibody (red); see S1 Data for underlying data. **** 0.0001; scale bars, 10 m. nt, nucleotide; PAM, protospacer adjacent motif.(TIF) pbio.3000557.s001.tif (1.6M) GUID:?8358B408-0973-471D-ADA8-02DC877569A7 S2 Fig: Characterization of altered Activin signaling in BMPR2-deficient ECs. (A) BMPR2-deficient ECs confer sensitivity to Activin A. Dose response (1.5, 3, 10 nM) of Activin ACdependent 4-Aminobenzoic acid phosphorylation of SMAD1/5 and SMAD2 upon 15 min of stimulation. si, small interfering(TIF) pbio.3000557.s002.tif (255K) GUID:?205F104B-0F08-4516-8E7F-9724C7804177 S3 Fig: BMPR2-deficient ECs signal through hetero-oligomers comprising BMP and TGF receptors as indicated by the formation of mixed SMAD complexes. (A) Immunoblot demonstrating efficiency OCTS3 of TR2 knock-down by siRNA (20 nM). (B) The ALK5 selective inhibitor SB-431542 abolishes BMP6-SMAD2 but not SMAD1/5 phosphorylation (upper), while the ALK2 selective inhibitor “type”:”entrez-nucleotide”,”attrs”:”text”:”K02288″,”term_id”:”191391″K02288 abolishes BMP6-SMAD1/5 phosphorylation (lower). (C) Epifluorescence images of PLA (left) showing complexes of SMAD5 (S5) with SMAD2/3 (S2/3) in indicated cell clones upon TGF stimulation (200 pM) for 15 min. PLA signals are pseudo-colored greyscale and inverted (upper). Scale bar, 10 m. (D) Quantification of SMAD5-SMAD2/3 PLA signals (right) in TGF-stimulated cells with the number of nuclear, cytosolic, and overall PLA foci shown. Data are presented as mean SD ( 7 frames, 20C30 cells each). See S2 Data for underlying data. (E) PLA controls for mutant ECs shown in panel C, 4-Aminobenzoic acid i.e., SMAD5 and SMAD2/3 antibodies alone (upper) or for PLA shown in Fig2E, i.e., SMAD1, SMAD2 antibodies alone (lower). (F) PLA positive control: 15 min 4-Aminobenzoic acid TGF (200 pM) stimulation for SMAD2/3-co-SMAD4 complexes in cells. Statistical significance relative to BMPR2wt was calculated using one-way ANOVA and Bonferroni post hoc test for PLA data; * 0.05, ** 0.01, *** 0.001, **** 0.0001. n.s., not significant(TIF) pbio.3000557.s003.tif (2.7M) GUID:?B05E443D-E273-499B-A211-C046F7110912 S4 Fig: Differential expression of TGF pathway members and increased SMAD1 occupancy at ID3 promoter. (A, B) RNA-Seq analysis of WT and BMPR2-deficient ECs under steady-state conditions (= 3 independent replicates). (A) Hierarchical clustering of differentially expressed TGF pathway members. Heatmap color coding shows z-score of differentially regulated genes (red = high; blue = low). (B) Relative expression of ligands, TGF, and BMP type-1, type-2 and co-receptors under steady-state conditions shown with RPKM values. Note that ALK1 and ENG are both significantly reduced in BMPR2-deficient ECs. (C) Verification of increased ITGB1 expression in BMPR2-deficient ECs by qRT-PCR analysis (= 6). (D) IGV browser displays over the loci showing SMAD1/5 ChIP-Seq track of HUVECs treated with BMP9  and pSMAD1/5 ChIP-Seq track of MDA-MB-231 cells treated with TGF1 . ChIP-Seq data were retrieved from the GEO (“type”:”entrez-geo”,”attrs”:”text”:”GSM684747″,”term_id”:”684747″GSM684747, “type”:”entrez-geo”,”attrs”:”text”:”GSM2429820″,”term_id”:”2429820″GSM2429820). (E) SMAD1 occupancy at the ID3 promoter was validated by ChIP-qPCR in steady-state conditions. IPs are a representative experiment of two, and ChIP-qPCR was performed in triplicates shown with means + SD. (F) Verification of altered expression in BMPR2-deficient ECs by qRT-PCR analysis ( 4). Statistical significance relative to BMPR2wt was calculated for RPKM values using one-way ANOVA and Bonferroni post hoc test and for qRT-PCR data using the Kruskal-Wallis test with post hoc Dunn test; * 0.05, ** 0.01, *** 0.001, **** 0.001. See S3 Data for underlying data. n.s., not significant(TIF) pbio.3000557.s004.tif (1.2M) GUID:?87DD9218-2137-4E55-9610-EAFC215545A5 S5 Fig: EndMT and alterations in F-actin organization induce subcellular stiffening. (A) Maximum projection of confocal z-stacks showing cell junctions of indicated cell clones immuno-labelled with an anti-N-Cadherin (green) antibody. (B) Single 4-Aminobenzoic acid confocal z-planes (medial) showing cell junctions of indicated cell clones immuno-labelled with an anti–catenin.