Journal: Oncology Reports

Article Title: POSTN + fibroblast-secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling

doi: 10.3892/or.2026.9067

Figure Lengend Snippet: BMP4 activates BMPR2/Smad signaling to induce macrophage M2 polarization. (A) PCA analysis of macrophages were treated with sEV-derived CAF-S6 transfected with shNC and shPOSTN in three replicate times. (B) Venn-diagram of DEGs between shPOSTN sEV vs. CTRL and shPOSTN sEVs vs. shNC sEVs. (C) Heatmap showing the top DEGs in each group. (D) Volcano plot showing BMP4 downregulation in macrophages treated with shPOSTN sEVs vs. shNC sEVs. (E) GSEA analysis of hallmark pathways in macrophages treated with sEV-derived CAF-S6 transfected with either shPOSTN or shNC. (F) GO enrichment analysis was performed to identify pathways associated with the representative DEGs. (G) The bubble plot displays the top 20 markedly enriched KEGG pathways for the DEGs. (H) The mRNA expression of BMP4 in macrophages induced sEVs derived from CAF-S5/-S6 transfected with shNC and shPOSTN were analyzed by RT-qPCR. (I) The mRNA expression of TNF-α, IL-6, TGF-β and IL-10 in macrophages stimulated with BMP4 at concentrations of 0, 50 and 100 ng/ml. (J) The expression of CD163 and CD206 in macrophages treated with BMP4 at concentrations of 0, 100 ng/ml examined by flow cytometry. (K) The level of BMPR2, pSmad1/5/9, Smad 5 and Smad 9 in macrophages treated with BMP4 (100 ng/ml) for 48 h were examined by western blotting (n=3 per group). (L) The level of pSmad1/5/9, Smad5, Smad9, CD163 and CD206 in macrophages treatment with or without LDN193189 inhibitor (n=3 per group). Statistical significance was determined using a one-way ANOVA test, ns, not significant *P<0.05; **P<0.01; ****P<0.0001. Error bars represent the mean ± SEM. Source data for blotting assays, see . PCA, principal component analysis; sEV, small extracellular vesicles; sh, short hairpin; NC, negative control; DEGs, differentially expressed genes; POSTN, perostin; sEV, small extracellular vesicles; GSEA, gene set enrichment analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR; pSmad, phosphorylated Smad.

Article Snippet: The primary antibodies included POSTN antibody (cat. no. ab14041; 1:500; Abcam), CD9 (cat. no. ab236630; 1:1,000; Abcam), CD81 (cat. no. ab79559; 1:1,000; Abcam), Calnexin (10427-2-AP; 1:1,000, Proteintech, Wuhan, China), CD80 (cat. no. ab134120; 1:1,000; Abcam), CD86 (cat. no. abs115477; 1:1,000; Absin Bioscience), CD163 (sc-33715; 1:1,000, Santa Cruz Biotechnology), CD206 (cat. no. ab64693; 1:1,000; Abcam), BMPR2 (cat. no. abs147034; 1:1,000; Absin Bioscience), Phospho-Smad 1 (Ser463/465)/Smad5(Ser463/465)/Smad9(Ser465/467) (cat. no. 13820; 1:1,000; Cell Signaling Technology, Inc.), Smad5 (12534; 1:1,000; Cell Signaling Technology, Inc.), Smad9 (cat. no. abs131190; 1:1,000; Absin Bioscience) and GAPDH (cat. no. 10494-1-AP; 1:5,000; Proteintech Group, Inc.), followed by incubation with a HRP-conjugated Goat anti-Rabbit IgG (H+L) as the secondary antibody (cat. no. SA00001-2; 1:3,000; Proteintech Group, Inc.) for 2 h. After washing the membrane three times with 1X TBST, the protein bands were visualized using an ECL detection system.

Techniques: Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

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