Journal: Cell Death & Disease

Article Title: Loss of FAM60A disrupts Sin3/HDAC control of the Hippo signaling and promotes oncogenic YAP1 activation

doi: 10.1038/s41419-026-08778-y

Figure Lengend Snippet: A A schematic of a refined proteomics workflow employing Mass spectrometry for protein identification. B HEK293T cells were transfected with Halo-SIN3A, and the cell lysates were subjected to affinity purification with Halo magnetic beads. Purified protein complexes were identified by Mass Spectrometry and analyzed using QSPEC (QSPEC log2FC ≥ 1, QSPEC FDR < 0.05). The results were then visualized in a differential network using Cytoscape. C Halo-SIN3A or Halo plasmid alone was ectopically expressed in HEK293T cells, followed by Halo affinity purification. The Halo affinity-purified proteins were subjected to immunoblotting with anti-FAM60A, anti-HDAC1, anti-SUDS3, anti-RBBP7, anti-RBBP4, and anti-SIN3A antibodies. Total cell lysates were also analyzed by immunoblotting using the same antibodies and anti-GAPDH as a loading control. D Identified subunits of Sin3/HDAC complex were N-terminally Halo-tagged and ectopically expressed in HEK293 cells, followed by affinity purification and identified using Mass-spectrometry analysis (QSPEC log2FC ≥ 1, FDR < 0.05) and visualized in a heatmap.

Article Snippet: Antibodies used in this study were sourced as follows: RbAp46/RBBP7 (A22274), RBBP4 (A3645), and SIN3A (A21966) from ABclonal, USA; Halo-Tag from Promega, USA (G9211); SAP30 (27679-1-AP) and SUDS3 (25845-1-AP) from Proteintech, USA; FAM60A from MRC PPU, Dundee University, UK (Cat # S381C).

Techniques: Mass Spectrometry, Transfection, Affinity Purification, Magnetic Beads, Purification, Plasmid Preparation, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Loss of FAM60A disrupts Sin3/HDAC control of the Hippo signaling and promotes oncogenic YAP1 activation

doi: 10.1038/s41419-026-08778-y

Figure Lengend Snippet: A Schematic diagram illustrates various deletions in the FAM60A gene. B Halo-tagged FAM60A deletion mutants were transfected into HEK293T cells, labeled with Halo-Tag TMRDirect fluorescent ligand (red), and DNA was stained using Hoechst dye (blue). C Halo-FAM60A deletion mutants were expressed in HEK293T cells, purified, and analyzed via proteomics (QSPEC log2FC ≥ 1, FDR < 0.05). Results were displayed in a heatmap. D The interactions of full-length and truncated FAM60A with Sin3/HDAC complex core-subunits were visualized in a heatmap (grey indicates no interaction). E Heatmap showing FAM60A conserved motif-specific interactions with Sin3/HDAC complex core-subunits (Grey signify no interaction).

Article Snippet: Antibodies used in this study were sourced as follows: RbAp46/RBBP7 (A22274), RBBP4 (A3645), and SIN3A (A21966) from ABclonal, USA; Halo-Tag from Promega, USA (G9211); SAP30 (27679-1-AP) and SUDS3 (25845-1-AP) from Proteintech, USA; FAM60A from MRC PPU, Dundee University, UK (Cat # S381C).

Techniques: Transfection, Labeling, Staining, Purification

Journal: Cell Death & Disease

Article Title: Loss of FAM60A disrupts Sin3/HDAC control of the Hippo signaling and promotes oncogenic YAP1 activation

doi: 10.1038/s41419-026-08778-y

Figure Lengend Snippet: A Workflow for Halo-FAM60A-expressing HEK293 cell extract preparation, Halo-Tag protein purification, size exclusion chromatography, and AP-MS analysis. B Halo-FAM60A affinity-purified proteins were fractionated through size-exclusion chromatography and analyzed by LC-MS and visualized in a heatmap (Red and Black). C HDAC1-Halo or Halo-HDAC2, or Halo-SUDS3 was ectopically expressed in HEK293T cells. Halo affinity purification followed by mass spectrometry analysis showed that FAM60A and SIN3A copurified with HDAC1-Halo and Halo-SUDS3 but not with Halo-HDAC2. D – F HDAC1-Halo or Halo-HDAC2, or Halo-SUDS3 or Halo plasmid alone, was ectopically expressed in HEK293T cells. Halo affinity purification was performed, followed by immunoblot analysis using specific antibodies and GAPDH as a loading control. G Whole-cell lysates from HEK293 cells were subjected to immunoprecipitation using an anti-FAM60A antibody, followed by immunoblotting with antibodies against HDAC1, HDAC2, SUDS3, and Sin3A. H HDAC1 and SUDS3 were individually or combinatorially knocked out using CRISPR/Cas9. Subsequently, either the Halo tag alone or Halo-FAM60A was overexpressed, and protein complexes were purified by affinity purification. These complexes were then subjected to immunoblotting analysis and probed with various antibodies.

Article Snippet: Antibodies used in this study were sourced as follows: RbAp46/RBBP7 (A22274), RBBP4 (A3645), and SIN3A (A21966) from ABclonal, USA; Halo-Tag from Promega, USA (G9211); SAP30 (27679-1-AP) and SUDS3 (25845-1-AP) from Proteintech, USA; FAM60A from MRC PPU, Dundee University, UK (Cat # S381C).

Techniques: Expressing, Protein Purification, Size-exclusion Chromatography, Protein-Protein interactions, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Western Blot, Control, Immunoprecipitation, CRISPR, Purification

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) Quantitative RT-PCR and immunoblot analysis confirming efficient SIN3a overexpression at the mRNA and protein levels in human proliferating failed donor FD-PASMCs following adenoviral transduction either with an empty vector (Ad-Control) or an adenoviral vector encoding human SIN3a (Ad-SIN3a) for 48 hours. (B) RNA sequencing workflow and analysis in SIN3a-overexpressing PASMCs and controls. ( C ) Heatmap of the top 5,000 DEGs illustrates a broad reprogramming of gene expression, consistent with SIN3a’s role as a master transcriptional regulator. (D) GAGE pathway enrichment analysis of differentially expressed genes reveals significant enrichment of pathways associated with TGF-β signaling, epithelial–mesenchymal transition (EMT), hypoxia, and ROS stress responses in SIN3a-overexpressing PASMCs. Functional interaction network of enriched pathways reveals SIN3a-driven upregulation (green) and downregulation (red) of signaling cascades, prominently involving extracellular matrix (ECM) dynamics and cellular stress responses . (E) WikiPathways enrichment identifies diverse regulatory modules following SIN3a overexpression, including nuclear receptor signaling and apoptotic pathways, implicating a multifaceted transcriptional role. ( F ) Cross-comparison with human PAH RNAseq datasets identifies a shared set of dysregulated genes modulated by SIN3a. Gene Set Enrichment Analysis (GSEA) demonstrates significant upregulation of gene sets linked to TGF-β signaling, epithelial-to-mesenchymal transition (EMT), and hypoxia. (G-I) Functional annotation of overlapping genes reveals enrichment in mitochondrial dysfunction, HIF1α activation, and TNF receptor signaling, implicating SIN3a in core disease-associated networks.

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Transduction, Plasmid Preparation, Control, RNA Sequencing, Gene Expression, Functional Assay, Comparison, RNA sequencing, Activation Assay

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) ( A ) qRT-PCR analysis of SIN3a mRNA expression in PASMCs cultured under normoxic conditions or exposed to hypoxia (1% O₂) and/or TGF-β1 stimulation. ( B-C ) qRT-PCR analysis of TGFB1 and BMPR2 mRNA expression under hypoxic and/or TGF-β1 conditions in the presence or absence of SIN3a overexpression. ( D ) ENCODE ChIP-seq analysis demonstrating HIF-1α binding at promoter regions of TGF-β signaling components, including TGFBR1 and TGFB1. ( E ) qRT-PCR analysis of HIF-1α mRNA expression under hypoxia and/or TGF-β1 stimulation, with or without SIN3a overexpression. ( F ) Representative immunoblot analysis of HIF-1α and BMPR2 protein expression under identical experimental conditions. ( G ) Immunofluorescence staining showing nuclear localization of HIF-1α and phosphorylation of SMAD1/5/9 under hypoxic and TGF-β1 conditions, demonstrating attenuation of hypoxia-induced nuclear translocation by SIN3a. ( H-I ) qRT-PCR analysis of COL1A1, COL3A1, NOX4, and NFE2L2 mRNA expression in PASMCs exposed to hypoxia (1% O₂) and TGF-β1, with or without SIN3a overexpression. ( J ) Reactive oxygen species detection in PASMCs cultured under normoxic or hypoxic (1% O₂) conditions using CellROX reagent (5 μM) added during the final 30 min of treatment. Cells were fixed, and CellROX fluorescence intensity (red) was quantified as a measure of intracellular oxidative stress. Data are presented as mean ± SEM. P < 0.05, P < 0.01, P < 0.001.

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Over Expression, ChIP-sequencing, Binding Assay, Western Blot, Immunofluorescence, Staining, Phospho-proteomics, Translocation Assay, Fluorescence

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) SIN3a mRNA expression in aortic tissue from tamoxifen-induced SMMHC-CreERT2 SIN3a fl/fl mice (SIN3a SMC-/- ) mice and littermates SIN3a fl/fl control mice. (B) Representative co-immunofluorescence staining of SIN3a (red) and α-smooth muscle actin (α-SMA; green) in aortic sections of SIN3a SMC-/- mice. ( C) Right heart catheterization analysis showing right ventricular systolic pressure (RVSP; left panel) in SIN3a SMC-/- KO and littermate control mice under normoxic and Sugen/Hypoxia (SuHx) conditions for 21 days. RV hypertrophy was quantified using the Fulton index (RV/LV+S), right panel. (D) Quantification of cardiomyocyte cross area assessed by wheat germ agglutinin (WGA) staining in SIN3a SMC-/- KO and littermate control mice under normoxic and SuHx conditions. (E) qRT-PCR analysis of pro-hypertrophic gene markers ( ANP, BNP, BMHC ) in RV from SIN3a-deficient mice compared to littermates under normoxic and SuHx conditions. (F) Representative Masson’s trichrome staining of RV sections of SIN3a SMC-/- KO mice and littermate control mice under normoxic and SuHx conditions. (G) Fibrosis-related gene expression ( TGFB, COL1A1, COL3A1 ) was measured by qRT-PCR in RV tissues from SIN3a SMC-/- KO mice compared to littermates exposed to normoxic and SuHx conditions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Expressing, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Gene Expression

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) Representative co-immunofluorescence staining of α-smooth muscle actin (α-SMA; green) and SIN3a (red) in lung sections from littermate control and SIN3a SMC-/- mice under normoxic and SuHx conditions. (B) Quantification of pulmonary arterial medial wall thickness and vascular remodeling in SIN3a SMC-/- mice littermate control and SIN3a SMC-/- mice under normoxic and SuHx conditions, as assessed by hematoxylin and eosin staining. (C-D) qRT-PCR analysis of inflammatory ( TNFA ) and profibrotic markers (TGFB, COL1A1, and COL3A1) in lungs from littermates and SIN3a SMC-/- mice under to normoxic and SuHx conditions. (E) qRT-PCR analysis of oxidative stress–related genes ( SOD2, NOX4, NFE2L2 ) in lungs from controls littermates and SIN3a SMC-/- mice under normoxic and SuHx conditions. (F-G) qRT-PCR analysis of BMPR2 and HIF1A transcript levels in lungs of littermates and SIN3a SMC-/- mice under normoxic and SuHx conditions. (H) Representative immunoblot analysis of BMPR2, phospho-SMAD2/3, and phospho-SMAD1/5/9 protein levels in lungs from SIN3a SMC-/- lungs. (I-J) Densitometric quantification of immunoblot data showing SIN3a and BMPR2 protein levels normalized to β-actin, and phospho-SMAD2/3 (TGF-β signaling) and phospho-SMAD1/5/9 (BMP signaling) normalized to total SMAD2/3 and SMAD1, respectively. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: Global transcriptomic profiling of PAH-PASMCs following SIN3a overexpression and combined SIN3a with Sotatercept treatment . ( A ) Principal component analysis (PCA) of RNA-seq data showing distinct clustering of control, SIN3a-overexpressing, and SIN3a + sotatercept-treated PAH-PASMCs. ( B ) Heatmap of significantly differentially expressed genes (DEGs) across experimental conditions. ( C ) Unsupervised hierarchical clustering illustrating treatment-specific transcriptional signatures and regulation of genes associated with proliferative signaling, cellular homeostasis, stress response, and TGF-β signaling. ( D ) Gene set enrichment analysis (GSEA) reveals enrichment for hypoxia- and oxidative stress-related pathways. ( E ) Differential expression of BMPR2 downstream target genes ID1, ID2, and ID3 following SIN3a and SIN3a + Sotatercept treatment. ( F-G ) Gene Ontology (GO) enrichment analyses highlighting biological processes related to vascular remodeling, ECM organization, hypoxia response, oxidative stress, and PASMC proliferation. ( H ) FPKM-normalized expression levels of ID1, ID2, and ID3 across control, SIN3a-overexpressing, and SIN3a + Sotatercept-treated PAH-PASMCs.

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Over Expression, RNA Sequencing, Control, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) Schematic overview of the experimental design used to evaluate the therapeutic efficacy of Sotatercept in the SuHx-induced PAH conditional SIN3a SMC-/- KO mouse model. Tissues were harvested 35 days after initiation of treatment for molecular and histological analyses. ( B ) RVSP (left panel) and RV hypertrophy assessed by the Fulton Index (RV/[LV+S]; right panel) in vehicle- and Sotatercept-treated SIN3a SMC KO mice. Representative right ventricular sections stained with fluorescent WGA to assess cardiomyocyte cross-sectional area, with corresponding quantitative analysis shown to the right. qRT-PCR analysis of hypertrophic marker gene expression ( ANP, BNP, BMHC ) in RV tissue from the indicated groups. ( E ) Representative Masson’s trichrome-stained RV sections from vehicle- and Sotatercept-treated SIN3a SMC-/- KO mice. ( F ). qRT-PCR analysis of profibrotic gene expression ( TGFB, COL1A1, COL3A1 ) in RV tissue from the indicated groups. ( G ). Representative hematoxylin and eosin (H&E)-stained lung sections from the indicated mice, with quantification of pulmonary arterial medial wall thickness shown on the right from vehicle- and Sotatercept-treated SIN3a SMC-/- KO mice. ( H ). qRT-PCR analysis of profibrotic marker expression in lung tissue from vehicle- and Sotatercept-treated SIN3a SMC-/- KO mice. ( I-J ). RT-PCR analysis of pro-inflammatory cytokines ( IL6, TNFA ) and oxidative stress–related genes ( NOX4, SOD2, NFE2L2 ) in the lungs of Sotatercept-treated SIN3a SMC-/- KO mice compared with vehicle-treated controls. Data are presented as mean ±SEM; ns: not significant, * p<0.05, *** p < 0.001.

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Drug discovery, Staining, Quantitative RT-PCR, Marker, Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: ( A ) qRT-PCR analysis of ID1, ID2 , and ID3 mRNA expression in lungs from SIN3a SMC-/- KO lungs treated with Sotatercept compared to vehicle-treated controls. ( B ) qRT-PCR analysis of HIF1α and BMPR2 mRNA expression in lungs from SIN3a SMC-/- KO lungs treated with Sotatercept compared to vehicle-treated controls. ( C-D ) Representative immunoblot analysis ( C ) and corresponding densitometric quantification ( D ) of SIN3a, BMPR2, SMAD signaling components, and HIF-1α protein levels in lungs from vehicle- and Sotatercept- treated SIN3a SMC-/- KO mice. ( E ) Schematic model illustrating the proposed mechanism by which SIN3a loss of function promotes PAH features and how Sotatercept treatment counteracts SIN3a deficiency. Sotatercept represses TGF-β signaling, HIF-1α expression, pro-inflammatory and oxidative stress pathways, while restoring BMPR2 signaling and pathway balance. Restoration of BMPR2/TGF-β signaling by SIN3a and Sotatercept limits oxidative stress, inflammation, and extracellular matrix remodeling through inhibition of HIF-1α–dependent pathways. Schematic Created with BioRender.com .

Article Snippet: In vivo, smooth muscle–specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) Quantitative RT-PCR and immunoblot analysis confirming efficient SIN3a overexpression at the mRNA and protein levels in human proliferating failed donor FD-PASMCs following adenoviral transduction either with an empty vector (Ad-Control) or an adenoviral vector encoding human SIN3a (Ad-SIN3a) for 48 hours. (B) RNA sequencing workflow and analysis in SIN3a-overexpressing PASMCs and controls. ( C ) Heatmap of the top 5,000 DEGs illustrates a broad reprogramming of gene expression, consistent with SIN3a’s role as a master transcriptional regulator. (D) GAGE pathway enrichment analysis of differentially expressed genes reveals significant enrichment of pathways associated with TGF-β signaling, epithelial–mesenchymal transition (EMT), hypoxia, and ROS stress responses in SIN3a-overexpressing PASMCs. Functional interaction network of enriched pathways reveals SIN3a-driven upregulation (green) and downregulation (red) of signaling cascades, prominently involving extracellular matrix (ECM) dynamics and cellular stress responses . (E) WikiPathways enrichment identifies diverse regulatory modules following SIN3a overexpression, including nuclear receptor signaling and apoptotic pathways, implicating a multifaceted transcriptional role. ( F ) Cross-comparison with human PAH RNAseq datasets identifies a shared set of dysregulated genes modulated by SIN3a. Gene Set Enrichment Analysis (GSEA) demonstrates significant upregulation of gene sets linked to TGF-β signaling, epithelial-to-mesenchymal transition (EMT), and hypoxia. (G-I) Functional annotation of overlapping genes reveals enrichment in mitochondrial dysfunction, HIF1α activation, and TNF receptor signaling, implicating SIN3a in core disease-associated networks.

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Transduction, Plasmid Preparation, Control, RNA Sequencing, Gene Expression, Functional Assay, Comparison, RNA sequencing, Activation Assay

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) ( A ) qRT-PCR analysis of SIN3a mRNA expression in PASMCs cultured under normoxic conditions or exposed to hypoxia (1% O₂) and/or TGF-β1 stimulation. ( B-C ) qRT-PCR analysis of TGFB1 and BMPR2 mRNA expression under hypoxic and/or TGF-β1 conditions in the presence or absence of SIN3a overexpression. ( D ) ENCODE ChIP-seq analysis demonstrating HIF-1α binding at promoter regions of TGF-β signaling components, including TGFBR1 and TGFB1. ( E ) qRT-PCR analysis of HIF-1α mRNA expression under hypoxia and/or TGF-β1 stimulation, with or without SIN3a overexpression. ( F ) Representative immunoblot analysis of HIF-1α and BMPR2 protein expression under identical experimental conditions. ( G ) Immunofluorescence staining showing nuclear localization of HIF-1α and phosphorylation of SMAD1/5/9 under hypoxic and TGF-β1 conditions, demonstrating attenuation of hypoxia-induced nuclear translocation by SIN3a. ( H-I ) qRT-PCR analysis of COL1A1, COL3A1, NOX4, and NFE2L2 mRNA expression in PASMCs exposed to hypoxia (1% O₂) and TGF-β1, with or without SIN3a overexpression. ( J ) Reactive oxygen species detection in PASMCs cultured under normoxic or hypoxic (1% O₂) conditions using CellROX reagent (5 μM) added during the final 30 min of treatment. Cells were fixed, and CellROX fluorescence intensity (red) was quantified as a measure of intracellular oxidative stress. Data are presented as mean ± SEM. P < 0.05, P < 0.01, P < 0.001.

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Over Expression, ChIP-sequencing, Binding Assay, Western Blot, Immunofluorescence, Staining, Phospho-proteomics, Translocation Assay, Fluorescence

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) SIN3a mRNA expression in aortic tissue from tamoxifen-induced SMMHC-CreERT2 SIN3a fl/fl mice (SIN3a SMC-/- ) mice and littermates SIN3a fl/fl control mice. (B) Representative co-immunofluorescence staining of SIN3a (red) and α-smooth muscle actin (α-SMA; green) in aortic sections of SIN3a SMC-/- mice. ( C) Right heart catheterization analysis showing right ventricular systolic pressure (RVSP; left panel) in SIN3a SMC-/- KO and littermate control mice under normoxic and Sugen/Hypoxia (SuHx) conditions for 21 days. RV hypertrophy was quantified using the Fulton index (RV/LV+S), right panel. (D) Quantification of cardiomyocyte cross area assessed by wheat germ agglutinin (WGA) staining in SIN3a SMC-/- KO and littermate control mice under normoxic and SuHx conditions. (E) qRT-PCR analysis of pro-hypertrophic gene markers ( ANP, BNP, BMHC ) in RV from SIN3a-deficient mice compared to littermates under normoxic and SuHx conditions. (F) Representative Masson’s trichrome staining of RV sections of SIN3a SMC-/- KO mice and littermate control mice under normoxic and SuHx conditions. (G) Fibrosis-related gene expression ( TGFB, COL1A1, COL3A1 ) was measured by qRT-PCR in RV tissues from SIN3a SMC-/- KO mice compared to littermates exposed to normoxic and SuHx conditions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Expressing, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Gene Expression

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) Representative co-immunofluorescence staining of α-smooth muscle actin (α-SMA; green) and SIN3a (red) in lung sections from littermate control and SIN3a SMC-/- mice under normoxic and SuHx conditions. (B) Quantification of pulmonary arterial medial wall thickness and vascular remodeling in SIN3a SMC-/- mice littermate control and SIN3a SMC-/- mice under normoxic and SuHx conditions, as assessed by hematoxylin and eosin staining. (C-D) qRT-PCR analysis of inflammatory ( TNFA ) and profibrotic markers (TGFB, COL1A1, and COL3A1) in lungs from littermates and SIN3a SMC-/- mice under to normoxic and SuHx conditions. (E) qRT-PCR analysis of oxidative stress–related genes ( SOD2, NOX4, NFE2L2 ) in lungs from controls littermates and SIN3a SMC-/- mice under normoxic and SuHx conditions. (F-G) qRT-PCR analysis of BMPR2 and HIF1A transcript levels in lungs of littermates and SIN3a SMC-/- mice under normoxic and SuHx conditions. (H) Representative immunoblot analysis of BMPR2, phospho-SMAD2/3, and phospho-SMAD1/5/9 protein levels in lungs from SIN3a SMC-/- lungs. (I-J) Densitometric quantification of immunoblot data showing SIN3a and BMPR2 protein levels normalized to β-actin, and phospho-SMAD2/3 (TGF-β signaling) and phospho-SMAD1/5/9 (BMP signaling) normalized to total SMAD2/3 and SMAD1, respectively. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: Global transcriptomic profiling of PAH-PASMCs following SIN3a overexpression and combined SIN3a with Sotatercept treatment . ( A ) Principal component analysis (PCA) of RNA-seq data showing distinct clustering of control, SIN3a-overexpressing, and SIN3a + sotatercept-treated PAH-PASMCs. ( B ) Heatmap of significantly differentially expressed genes (DEGs) across experimental conditions. ( C ) Unsupervised hierarchical clustering illustrating treatment-specific transcriptional signatures and regulation of genes associated with proliferative signaling, cellular homeostasis, stress response, and TGF-β signaling. ( D ) Gene set enrichment analysis (GSEA) reveals enrichment for hypoxia- and oxidative stress-related pathways. ( E ) Differential expression of BMPR2 downstream target genes ID1, ID2, and ID3 following SIN3a and SIN3a + Sotatercept treatment. ( F-G ) Gene Ontology (GO) enrichment analyses highlighting biological processes related to vascular remodeling, ECM organization, hypoxia response, oxidative stress, and PASMC proliferation. ( H ) FPKM-normalized expression levels of ID1, ID2, and ID3 across control, SIN3a-overexpressing, and SIN3a + Sotatercept-treated PAH-PASMCs.

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Over Expression, RNA Sequencing, Control, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: (A) Schematic overview of the experimental design used to evaluate the therapeutic efficacy of Sotatercept in the SuHx-induced PAH conditional SIN3a SMC-/- KO mouse model. Tissues were harvested 35 days after initiation of treatment for molecular and histological analyses. ( B ) RVSP (left panel) and RV hypertrophy assessed by the Fulton Index (RV/[LV+S]; right panel) in vehicle- and Sotatercept-treated SIN3a SMC KO mice. Representative right ventricular sections stained with fluorescent WGA to assess cardiomyocyte cross-sectional area, with corresponding quantitative analysis shown to the right. qRT-PCR analysis of hypertrophic marker gene expression ( ANP, BNP, BMHC ) in RV tissue from the indicated groups. ( E ) Representative Masson’s trichrome-stained RV sections from vehicle- and Sotatercept-treated SIN3a SMC-/- KO mice. ( F ). qRT-PCR analysis of profibrotic gene expression ( TGFB, COL1A1, COL3A1 ) in RV tissue from the indicated groups. ( G ). Representative hematoxylin and eosin (H&E)-stained lung sections from the indicated mice, with quantification of pulmonary arterial medial wall thickness shown on the right from vehicle- and Sotatercept-treated SIN3a SMC-/- KO mice. ( H ). qRT-PCR analysis of profibrotic marker expression in lung tissue from vehicle- and Sotatercept-treated SIN3a SMC-/- KO mice. ( I-J ). RT-PCR analysis of pro-inflammatory cytokines ( IL6, TNFA ) and oxidative stress–related genes ( NOX4, SOD2, NFE2L2 ) in the lungs of Sotatercept-treated SIN3a SMC-/- KO mice compared with vehicle-treated controls. Data are presented as mean ±SEM; ns: not significant, * p<0.05, *** p < 0.001.

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Drug discovery, Staining, Quantitative RT-PCR, Marker, Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

doi: 10.64898/2026.02.03.703590

Figure Lengend Snippet: ( A ) qRT-PCR analysis of ID1, ID2 , and ID3 mRNA expression in lungs from SIN3a SMC-/- KO lungs treated with Sotatercept compared to vehicle-treated controls. ( B ) qRT-PCR analysis of HIF1α and BMPR2 mRNA expression in lungs from SIN3a SMC-/- KO lungs treated with Sotatercept compared to vehicle-treated controls. ( C-D ) Representative immunoblot analysis ( C ) and corresponding densitometric quantification ( D ) of SIN3a, BMPR2, SMAD signaling components, and HIF-1α protein levels in lungs from vehicle- and Sotatercept- treated SIN3a SMC-/- KO mice. ( E ) Schematic model illustrating the proposed mechanism by which SIN3a loss of function promotes PAH features and how Sotatercept treatment counteracts SIN3a deficiency. Sotatercept represses TGF-β signaling, HIF-1α expression, pro-inflammatory and oxidative stress pathways, while restoring BMPR2 signaling and pathway balance. Restoration of BMPR2/TGF-β signaling by SIN3a and Sotatercept limits oxidative stress, inflammation, and extracellular matrix remodeling through inhibition of HIF-1α–dependent pathways. Schematic Created with BioRender.com .

Article Snippet: We generated smooth muscle cell-specific SIN3a knockout mice (SIN3a SMC-/- ) and subjected them to the Sugen/hypoxia protocol to induce PAH.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Inhibition