Journal: Gastroenterology

Article Title: BMPR2 as a Novel Predisposition Gene for Hereditary Colorectal Polyposis.

doi: 10.1053/j.gastro.2023.03.006

Figure Lengend Snippet: Figure 1. The BMP pathway in the colon. BMP2 and BMP4 are the most abundant ligands of the BMP pathway in the bowel mucosa. They are able to bind the BMPR1A-BMPR2 complex on the surface of colonocytes, therefore, triggering R-SMAD phosphorylation and downstream gene regulation in the canonical pathway. In addition, BMP can also mediate proliferation by convergent pathways, such as ERK, JNK, and p38. Created with BioRender.com.

Article Snippet: The identified BMPR2 variants were introduced into the BMPR2 coding sequence (no. 116718, Addgene) by a simple 1- step PCR amplification with nonoverlapping primers (Q5 SiteDirected Mutagenesis Kit, New England Biolabs, Ipswich, MA).

Techniques: Phospho-proteomics

Journal: Gastroenterology

Article Title: BMPR2 as a Novel Predisposition Gene for Hereditary Colorectal Polyposis.

doi: 10.1053/j.gastro.2023.03.006

Figure Lengend Snippet: Figure 2. BMPR2 variants drive a proliferative response and impair the BMP4-mediated growth inhibition. Characterization of the proliferative capacity of 2 different BMPR2 clones (B26 and B31) in which the 3 BMPR2 variants were reintroduced. (A) MTS cell proliferation assay. Samples were assayed in triplicate and the experiment was repeated 5 times (n ¼ 5). (B) Plating efficiency (colonies originated from single cells) in the colony-formation assay. Samples were assayed in duplicate and the experiment was repeated 3 times (n ¼ 3). (C) MTS cell proliferation assay in the presence or absence of 50 ng/mL of BMP4. Data are represented as the growth difference between treated and untreated cells. Samples were assayed in triplicate and the experiment was repeated 4 times (n ¼ 4). Data represent mean ± SD. *P < .05, **P < .01, ***P < .001 for the analysis of variance with the least significant difference post-hoc test.

Article Snippet: The identified BMPR2 variants were introduced into the BMPR2 coding sequence (no. 116718, Addgene) by a simple 1- step PCR amplification with nonoverlapping primers (Q5 SiteDirected Mutagenesis Kit, New England Biolabs, Ipswich, MA).

Techniques: Inhibition, Clone Assay, Proliferation Assay, Colony Assay

Journal: Gastroenterology

Article Title: BMPR2 as a Novel Predisposition Gene for Hereditary Colorectal Polyposis.

doi: 10.1053/j.gastro.2023.03.006

Figure Lengend Snippet: Figure 3. BMPR2 variants act through SMAD or non-SMAD effectors, depending on their position. Characterization of the SMAD1/5/8 pathway activity of 2 different BMPR2 clones (B26 and B31) in which the 3 BMPR2 variants were reintroduced. (A) Quantitative analysis of the phosphorylated levels of SMAD1 (Ser463/465) by means of enzyme-linked immunosorbent assay in the presence or absence of 50 ng/mL of BMP4. Samples were assayed in duplicate and the experiment was repeated 3 times (n ¼ 3). (B) Real-time PCR quantification of ID1 and ID3 expression levels, 2 canonical BMP-SMAD downstream targets, in clone B26. Samples were assayed in triplicate and the experiment was repeated 3 times (n ¼ 3). Data represent mean ± SD. *P < .05, **P < .01, ***P < .001 for the analysis of variance with the least significant difference post-hoc test.

Article Snippet: The identified BMPR2 variants were introduced into the BMPR2 coding sequence (no. 116718, Addgene) by a simple 1- step PCR amplification with nonoverlapping primers (Q5 SiteDirected Mutagenesis Kit, New England Biolabs, Ipswich, MA).

Techniques: Activity Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: European Journal of Histochemistry : EJH

Article Title: Expression of bone morphogenetic protein signaling pathway players in the jejunum and colon of adult rats

doi: 10.4081/ejh.2025.4174

Figure Lengend Snippet: Immunohistochemical localization of ACVR1 ( a,b,c ) and BMPR2 ( d,e,f ) receptors in rat jejunum. e ) The arrows show strong immunostaining in the lamina propria; the asterisk designates the longitudinal muscularis layer. To determine the localization of BMP receptors, we performed immunohistochemical staining on jejunum sections from 6 different rats. Sections were examined by light microscopy (Leica DM 2000) using 20x and 40x objectives. Representative images are shown. Scale bars: 50 μm.

Article Snippet: BMPR2 , 1:100 , ProteinTech - 19087-1 - AP.

Techniques: Immunohistochemical staining, Immunostaining, Staining, Light Microscopy

Journal: European Journal of Histochemistry : EJH

Article Title: Expression of bone morphogenetic protein signaling pathway players in the jejunum and colon of adult rats

doi: 10.4081/ejh.2025.4174

Figure Lengend Snippet: Immunohistochemical localization of ACVR1 ( a,b,c ) and BMPR2 ( d,e,f ) receptors in rat colon. b ) The asterisk shows surface colonocytes. To determine the localization of BMP receptors, we performed immunohistochemical staining on colon sections from 6 different rats. Sections were examined by light microscopy (Leica DM 2000) using 20x and 40x objectives. Representative images are shown. Scale bars: 50 μm.

Article Snippet: BMPR2 , 1:100 , ProteinTech - 19087-1 - AP.

Techniques: Immunohistochemical staining, Staining, Light Microscopy

Journal: Scientific Reports

Article Title: Vitamin D receptor and its antiproliferative effect in human pulmonary arterial hypertension

doi: 10.1038/s41598-024-78380-9

Figure Lengend Snippet: Calcitriol inhibits PASMC proliferation via BMPR2. Human control PASMC were transfected with siRNA for BMPR2 (siBMPR2) or control siRNA (scramble). ( A ) BMPR2 mRNA expression assessed by qRT-PCR after 48 h post-transfection. Data is expressed as scatter plots and bars with medians, * p < 0.05, Mann–Whitney test. ( B ) Effects of calcitriol on proliferation in silenced BMPR2 PASMC measured by MTT assay. Results are expressed as mean ± SEM. *p < 0.05, t-test vs scramble. ( C ) and ( D ) Effects of calcitriol (1–100 nmol/l) on proliferation in PASMC transfected with siBMPR2 or scramble, measured by MTT and BrdU assays, respectively. * p < 0.05, two-way ANOVA, Bonferroni post hoc test, vs scramble. ( E ) Effects of calcitriol on proliferation in PASMC in the presence or absence of DMH1 (5 µmol/l), by MTT and BrdU assay; *** p < 0.001 calcitriol vs vehicle (black column), two way-ANOVA. n = 4–5 different cultures in duplicate or triplicate.

Article Snippet: Human control PASMC were transfected with specific siRNA against BMPR2 (SR300456B, OriGene, Maryland, USA), KCNK3 (gene encoding TASK-1, SASI_Hs01_00108786, Sigma-Aldrich, St. Louis, USA), BIRC5 ( gene encoding survivin, SASI_Hs01_00052229, Sigma-Aldrich, St. Louis, USA), and control non-targeting siRNA (SIC001 scramble siRNA, Sigma-Aldrich, St. Louis, USA), using LipofectamineTM RNAiMAX (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions as previously reported .

Techniques: Control, Transfection, Expressing, Quantitative RT-PCR, MANN-WHITNEY, MTT Assay, BrdU Staining

Journal: Cell

Article Title: Therapeutic potential of allosteric HECT E3 ligase inhibition

doi: 10.1016/j.cell.2025.03.001

Figure Lengend Snippet: Structural details of inhibitor binding to SMURF1, conservation of lysine residues on α helix #1, and AlphaFold model of the SMURF1:BMPR2 complex, related to , , , and (A) Crystallography parameters. ∗ The highest resolution shell is shown in parenthesis. (B) Surface representation of SMURF1 shows that the inhibitor is minimally exposed. (C) Detailed structural insight into the SMURF1—inhibitor complex, with a zoomed view of the binding cavity from three different angles. (D) 2D representation of the inhibitor binding site, rendered using LigPlot+ v.2.2 https://www.ebi.ac.uk/thornton-srv/software/LigPlus/ . (E) mFo-DFc simulated-annealing electron density omit map showing the α10 and the hinge region without (left) and with (right) the inhibitor. The map was calculated by omitting the entire model of the α 10, the hinge, and the inhibitor using simulated-annealing sigma A analysis contoured at 2.75 (SMURF1, apo) and 2.05 Å (SMURF1, Cpd-8) at 3σ. (F) Structures of HECT ligase showing conservation of lysine residues on α helix #1. Superimposing the structures of Rsp5 (3OLM), NEDD4 (2XBB), and SMURF1 and AlphaFold model of E6AP/UBE3A HECT domains shows the conservation of lysine (K) residues on α helix #1, previously demonstrated to undergo self-ubiquitylation that downregulates the ligase activity. (G) AlphaFold model of the SMURF1:BMPR2 complex. The sequences of full-length SMURF1 and the intracellular domain of BMPR2 were modeled in AlphaFold3. The domains of SMURF1 are indicated. Residues predicted to participate in binding are shown as ball-and-sticks. The model suggests that WW1 and WW2 domains directly interact with the kinase domain of BMPR2.

Article Snippet: The following primary antibodies were used and paired with appropriate secondary antibodies for detection: SMURF1 (H00057154-M01, Abnova, USA and H60, Sant Cruz Biotechnology, USA), BMPR2 (orb69398, Biorbyt, UK and BD, #612292), anti-myc (sc-40, Sant Cruz Biotechnology, USA), SMAD1/5/9 (ab66737, Abcam, USA), phospho-SMAD1/5 (9516S, Cell Signaling Technology, USA), ID1 (M085, CalBioreagents, USA), GAPDH (2118L, Cell signaling Technology, USA), Fla-Tag (FG4R, Thermo Fisher Scientific, MA1-91878), Pan Actin (4968S, Cell Signaling) and beta-actin (4967, Cell Signaling Technology, USA).

Techniques: Binding Assay, Software, Activity Assay

Journal: Cell

Article Title: Therapeutic potential of allosteric HECT E3 ligase inhibition

doi: 10.1016/j.cell.2025.03.001

Figure Lengend Snippet: SMURF1 inhibitors restore BMP signaling and pulmonary vascular cell homeostasis (A) Scheme of SMURF1-BMPR2 and SMAD1 split-CAT based E. coli selection system, showing target ubiquitylation resulting in CAT assembly, resistance, and selective growth. (B and C) Hyperactive K381R (black) increases and catalytically inactive C725A (red) reduces SMURF1-dependent direct target ubiquitylation of SMAD1 (B) and BMPR2 (C) (inset represents area-under-the-curve of relative growth; n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s correction). (D) Representative western blot demonstrating stabilization of overexpressed SMAD1 in HEK cells in the presence of SMURF1 inhibitor. Mutations that reduce the flexibility (G634P) and length (Δ637 KID ) of the hinge result in reduce SMURF1 activity. Mutations that preserve the flexibility (G633C, D636G [GGLD]) and length (637GGLD INS ) of the SMURF1 glycine hinge escape the effect of the inhibitor. (E) Representative western blot demonstrating stabilization of overexpressed BMPR2-myc in HEK cells in the presence of SMURF1 inhibitor. (F) Immunoblotting of BMPR2, SMURF1, SMAD1/5/8, phosphorylated SMAD1/5/8, ID1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in PASMC cultured without or with BMP4 and SMURF1 inhibitor (Cpd-6) ( n = 9 separate experiments across cells from 3 PAH donor lungs, mean ± SEM). (G and H) Quantification of apoptosis in PAECs. (G) Representative time curve ( n = 5 technical replicates, mean ± SD) and (H) group data at 300 min with Cpd-6 or vehicle ( n = 6 separate donors, mean ± SEM). (I and J) Quantification of proliferation: (I) representative time course ( n = 5 technical replicates, mean ± SD) in PAEC with cell confluence mask for each dose of Cpd-6 or vehicle (orange) and (J) group data ( n = 3 separate PAEC donors; mean ± SEM). (K) Migration of PASMCs with Cpd-6 or vehicle measured via disc closure assay ( n = 2–3 separate donors). (L) Representative time course plots showing proliferation of PASMC from an idiopathic PAH patient with Cpd-6 or vehicle with cell confluence mask for each dose (orange) ( n = 5 technical replicates per concentration from one idiopathic PAH donor line, mean ± SD). (M) Group proliferation data at 72 h in PASMCs from patients with idiopathic or hereditary PAH ( n = 4–5 separate donor lines, mean ± SEM). ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunnett’s correction. See also , , and .

Article Snippet: The following primary antibodies were used and paired with appropriate secondary antibodies for detection: SMURF1 (H00057154-M01, Abnova, USA and H60, Sant Cruz Biotechnology, USA), BMPR2 (orb69398, Biorbyt, UK and BD, #612292), anti-myc (sc-40, Sant Cruz Biotechnology, USA), SMAD1/5/9 (ab66737, Abcam, USA), phospho-SMAD1/5 (9516S, Cell Signaling Technology, USA), ID1 (M085, CalBioreagents, USA), GAPDH (2118L, Cell signaling Technology, USA), Fla-Tag (FG4R, Thermo Fisher Scientific, MA1-91878), Pan Actin (4968S, Cell Signaling) and beta-actin (4967, Cell Signaling Technology, USA).

Techniques: Selection, Western Blot, Activity Assay, Cell Culture, Migration, Concentration Assay

Journal: Cell

Article Title: Therapeutic potential of allosteric HECT E3 ligase inhibition

doi: 10.1016/j.cell.2025.03.001

Figure Lengend Snippet: SMURF1 expression in PAH (A) Schematic representation of canonical BMP signaling resulting in SMAD1/5/8 phosphorylation, in nuclear translocation of SMAD4 and ID1 expression, and its negative regulation by SMURF1-mediated ubiquitylation and degradation of BMPR2 and SMAD1/5/8. (B) In HEK293 cells stably transfected with GFP-tagged SMURF1, BMP4 stimulation results in a decrease in GFP signal and short interfering RNA (siRNA) knockdown of ACVRL1, BMPR2, Endoglin (ENG), or SMAD9, and BMP4 stimulation results in increased GFP signal. n = 3 separate experiments; presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, relative to untreated, unpaired Student's t test. (C) Expression of SMURF1 is increased in PASMC from patients with idiopathic and heritable PAH when compared with PASMC from patients without PAH. n = 6 PAH donor lines and n = 9 control lines; presented as mean ± SEM. ∗∗ p < 0.01, unpaired Student's t test. (D) SMURF1 expression in the pulmonary artery intima and media in patients with PAH. SMURF1 (purple) protein co-localization with an endothelial (von Willebrand factor [vWF], yellow) or smooth muscle marker (Alpha Smooth Muscle Actin [αSMA], yellow) is indicated by a red/brown color shift. Representative images were obtained from 19 controls and 33 patients with PAH. Scale bar, 60 μm. Arrows indicate areas of co-localization. See also .

Article Snippet: The following primary antibodies were used and paired with appropriate secondary antibodies for detection: SMURF1 (H00057154-M01, Abnova, USA and H60, Sant Cruz Biotechnology, USA), BMPR2 (orb69398, Biorbyt, UK and BD, #612292), anti-myc (sc-40, Sant Cruz Biotechnology, USA), SMAD1/5/9 (ab66737, Abcam, USA), phospho-SMAD1/5 (9516S, Cell Signaling Technology, USA), ID1 (M085, CalBioreagents, USA), GAPDH (2118L, Cell signaling Technology, USA), Fla-Tag (FG4R, Thermo Fisher Scientific, MA1-91878), Pan Actin (4968S, Cell Signaling) and beta-actin (4967, Cell Signaling Technology, USA).

Techniques: Expressing, Phospho-proteomics, Translocation Assay, Stable Transfection, Transfection, Small Interfering RNA, Knockdown, Control, Marker

Journal: Cell

Article Title: Therapeutic potential of allosteric HECT E3 ligase inhibition

doi: 10.1016/j.cell.2025.03.001

Figure Lengend Snippet: BMP signaling and proteomic effects of SMURF1 inhibition in mammalian cells, related to (A) Comparison of small-molecule SMURF1 inhibition and siRNA-mediated SMURF1 knockdown on BMP signaling (BMP response element – ID1 promoter activation) in HEK293 cells. (B–D) Summary of expression proteomics experiments comparing PASMCs treated under hypoxic conditions ±BMP4 or ±SMURF1 inhibitor Cpd-6 (SMURF1i). (B) Experimental workflow. Table: sample conditions for PASMC BMPR2 (C347Y) mutant cells undergoing TMT quantitative proteomics profiling at 24 h. (C) Log 2 ratios of protein abundances for treated versus control (DMSO) in selected conditions. Data represent two biological replicates ( n = 2) per treatment condition in a single experiment derived from individual donors. Highlighted proteins correspond to “IL-1 beta- and endothelin-1-induced fibroblast/myofibroblast migration and extracellular matrix production in asthmatic airways” gene set, which was significantly enriched among dysregulated proteins, only in condition (B) (ii). Inserts show heatmap for ratios for donors 1 and 2 for proteins highlighted in the plot. (D) Log 2 ratio of known SMURF1 targets RhoA and TGFBR1 detected in proteomic study and chemiluminescence of SMURF1, BMPR2, SMAD1, pSMAD1, and ID1 in samples used for proteomic studies measured by western (not detected by proteomics). (E) Summary of significantly enriched terms for mutant cells under hypoxic conditions pre-treated with BMP4, followed by treatment with SMURF1 inhibitor or DMSO.

Article Snippet: The following primary antibodies were used and paired with appropriate secondary antibodies for detection: SMURF1 (H00057154-M01, Abnova, USA and H60, Sant Cruz Biotechnology, USA), BMPR2 (orb69398, Biorbyt, UK and BD, #612292), anti-myc (sc-40, Sant Cruz Biotechnology, USA), SMAD1/5/9 (ab66737, Abcam, USA), phospho-SMAD1/5 (9516S, Cell Signaling Technology, USA), ID1 (M085, CalBioreagents, USA), GAPDH (2118L, Cell signaling Technology, USA), Fla-Tag (FG4R, Thermo Fisher Scientific, MA1-91878), Pan Actin (4968S, Cell Signaling) and beta-actin (4967, Cell Signaling Technology, USA).

Techniques: Inhibition, Comparison, Knockdown, Activation Assay, Expressing, Mutagenesis, Quantitative Proteomics, Control, Derivative Assay, Migration, Western Blot

Journal: Cell Death Discovery

Article Title: BMP signaling is a therapeutic target in ovarian cancer

doi: 10.1038/s41420-020-00377-w

Figure Lengend Snippet: a Expression of mRNAs for BMP ligands and receptors in 306 OC patients. RNA-seq of TCGA serous ovarian cancer dataset was analyzed via cBioPortal. RNA expression cutoff Z score was adjusted to 2.0. b Correlation between BMPR2 mRNA expression and overall survival of 306 OC patients derived from the TCGA serous ovarian cancer dataset. Based on BMPR2 mRNA expression, the 306 patients were equally divided into three groups (high, middle, low). A P -value was calculated with a log-rank test. c BMPR2 protein expression was evaluated by immunoblotting (IB) in 6 OC cell lines. d SKOV3 cells were transfected with an empty (CT) or BMPR2-encoding plasmid for 72 h. The expression of indicated proteins was assessed by IB. e SKOV3 cells were treated with a Negative Control siRNA (siNC) and three different siBMPR2 for 72 h. The expression of indicated proteins was evaluated by IB. f ID3 mRNA expression of SKOV3 cells transfected with CT and BMPR2 plasmid for 72 h was analyzed by RT-PCR. g ID3 mRNA expression of SKOV3 cells treated with siNC and three different siBMPR2 for 72 h was assessed by RT-PCR. h SKOV3 and OVSAHO cells were transfected with CT or BMPR2 plasmid for 48 h, and thereafter cells were plated in 96-well plates and incubated for an additional 48 h. Cell viability was determined by MTS assay after adjusting CT to 1. i After 48 h treatment with siNC or three different BMPR2 siRNAs, SKOV3 and OVSAHO cells were cultured in 96-well plates for 48 h. MTS assay was used to assess cell viability relative to siNC. j , k Six ovarian cancer cells were treated with DMSO or different concentrations of LDN193189 ( j ) or RK783 ( k ) for 72 h. MTS assay was used to analyze cell numbers relative to DMSO treatment. l , m SKOV3 cells were incubated with DMSO or different concentrations of LDN193189 (LDN) ( l ) or RK783 (RK) ( m ) for 24 h. IB was used to analyze the expression of indicated proteins. The results in ( f – i ) are shown as the mean ± SE.

Article Snippet: BMPR2 expression plasmid was purchased from Addgene (Cambridge, MA, UK) and transfected into OC cells using Lipofectamine 2000 transfection reagent (Invitrogen). pcDNA 3.0 (Invitrogen) was used as a control (CT).

Techniques: Expressing, RNA Sequencing, RNA Expression, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Incubation, MTS Assay, Cell Culture

Journal: Genes

Article Title: Reduction of BMPR2 mRNA Expression in Peripheral Blood of Pulmonary Arterial Hypertension Patients: A Marker for Disease Severity?

doi: 10.3390/genes13050759

Figure Lengend Snippet: Baseline characteristics of PAH patients.

Article Snippet: Serum from each proband was pipetted into a 96-well plate and treated with ELISA reagents according to the manufacturer’s instructions (human BMPR2 ELISA kit, Elabscience, Wuhan, China).

Techniques: Variant Assay, Biomarker Discovery, Filtration, Diffusion-based Assay

Journal: Genes

Article Title: Reduction of BMPR2 mRNA Expression in Peripheral Blood of Pulmonary Arterial Hypertension Patients: A Marker for Disease Severity?

doi: 10.3390/genes13050759

Figure Lengend Snippet: Recruitment of study population from May 2019 to January 2020. 110 participants were screened, and samples were collected of which 109 were analyzed and included. BMPR2 : bone morphogenetic protein receptor type II, PAH: pulmonary arterial hypertension, PVOD: pulmonary veno-occlusive disease.

Article Snippet: Serum from each proband was pipetted into a 96-well plate and treated with ELISA reagents according to the manufacturer’s instructions (human BMPR2 ELISA kit, Elabscience, Wuhan, China).

Techniques:

Journal: Genes

Article Title: Reduction of BMPR2 mRNA Expression in Peripheral Blood of Pulmonary Arterial Hypertension Patients: A Marker for Disease Severity?

doi: 10.3390/genes13050759

Figure Lengend Snippet: Mean relative BMPR2 mRNA expression in whole blood. The boxplots provide median values (horizontal lines), interquartile range (box), 1.5× interquartile range (whiskers), outliers (indicated by circles within 1.5 to 3× interquartile range) and extreme outliers (indicated by asterisks > 3× interquartile range). A significant difference of BMPR2 mRNA expression could be identified between healthy controls, BMPR2 non-carriers and BMPR2 variant carriers. With Bonferroni correction, p -values were healthy controls vs. non-carriers p = 0.453, non-carriers vs. variant carriers p = 0.0002, and healthy controls vs. variant carriers p < 0.0001. The 1/delta cycle threshold (1/∆CT) denotes the level of BMPR2 mRNA gene expression measured by qPCR. n.s. = non-significant.

Article Snippet: Serum from each proband was pipetted into a 96-well plate and treated with ELISA reagents according to the manufacturer’s instructions (human BMPR2 ELISA kit, Elabscience, Wuhan, China).

Techniques: Expressing, Variant Assay, Gene Expression

Journal: Genes

Article Title: Reduction of BMPR2 mRNA Expression in Peripheral Blood of Pulmonary Arterial Hypertension Patients: A Marker for Disease Severity?

doi: 10.3390/genes13050759

Figure Lengend Snippet: Correlation of BMPR2 mRNA expression with laboratory and clinical characteristics.

Article Snippet: Serum from each proband was pipetted into a 96-well plate and treated with ELISA reagents according to the manufacturer’s instructions (human BMPR2 ELISA kit, Elabscience, Wuhan, China).

Techniques: Expressing, Filtration