cnn1  (Proteintech)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Aging‐Associated Nox4 ‐Mediated Mitochondrial Reactive Oxygen Species and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1161/JAHA.125.044949

Figure Lengend Snippet: A , B , Flow cytometry analysis of single‐cell suspension from the abdominal aorta of mice treated with Ang II. A , presents the quantification of MYH11 + cells; B , shows the quantification of CD45 + CD68 + CD11b + cells. The data represent the cell fraction of all aortic cells (mean±SEM, n=6). C, Flow cytometry analysis and quantification of abdominal aorta single‐cell suspension from Ang II‐treated mice showing the proportion of MYH11 + cells expressing macrophage markers. These data show the fraction of CD68 + CD11b + cells as a percentage of MYH11 + cells (mean±SEM, n=6). D , A t‐SNE clustering analysis of flow cytometry data from the abdominal aorta of Ang II‐treated mice was performed. MYH11 + cells were clustered based on the expression of CNN1, ACTA2, TAGLN, CD45, CD68, CD11b, CD38, TNFα, IL1b, and IL6, resulting in distinct clusters presented on the t‐SNE plot. E , A heat map representation illustrates the relative expression of SMC, macrophage, and inflammatory markers based on the mean fluorescence intensity for each cluster. F – H , The relative abundance of distinct clusters as a proportion of MYH11 + cells (mean±SEM, n=6). ACTA2 indicates actin alpha 2; Ang II, angiotensin II; Apoe −/− , apolipoprotein E knockout mice; MYH11, myosin heavy chain 11; cGAS, cyclic GMP‐AMP synthase; CNN, Calponin 1; IL‐1b, interleukin 1 beta; IL‐6, interleukin 6; MYH11, myosin heavy chain 11; Nox4 TG, Nox4 transgenic mice; Nox4 −/− , Nox4 knockout mice; SMC, smooth muscle cell; STING, stimulator of interferon gene; TAGLN, transgelin; TNFα, tumor necrosis factor alpha; t‐SNE, t‐distributed stochastic neighbor embedding; and TUBB, tubulin beta.

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: MYH11‐FITC (Biorbyt, Durham, NC; orb13894), CNN1 (calponin‐1)‐AlexaFluor 555 (Bioss; bs‐0095R‐A555), ACTA2‐DyLight 405, TAGLN‐PerCP (Novus Biologicals, Centennial, CO; NBP2‐34522V, NBP2‐47689PCP), TNFα (tumor necrosis factor alpha)‐Brilliant Violet 510 (BioLegend; 506 339), IL1b‐APC‐Cy7 (LifeSpan Bio, Newark, CA; LS‐C730762‐100), and IL6‐PerCP‐eFluor 710 (Thermo Fisher; 46–7061‐80).

Techniques: Flow Cytometry, Single Cell, Suspension, Expressing, Fluorescence, Knock-Out, Transgenic Assay

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A-C) Bar plots showing differential regulation of pericyte markers (RGS5, ABCC9), VSMC markers (CNN1, MYH11), and pan-mural cell markers (ACTA2, SMTN, TAGLN) in fibroblasts treated with (A) NGF, (B) BDNF, or (C) NT3 compared with untreated fibroblasts. Bar heights represent log₂ fold change, and bar color indicates the corresponding P values. (D) Schematic of the collagen gel contraction assay in which synovial fibroblasts were embedded in collagen I gels and treatment (E) Representative images of collagen I gel contraction following treatment with NGF, BDNF, or NT3 (F) Quantification of collagen I gel contraction expressed as percent contraction. Values represent mean ± standard deviation (SD). Individual data points indicate biological replicates. Statistical analysis was performed using one-way ANOVA, with P values shown. (G) Schematic illustrating the synovial organoid/explant experimental design. (H) Representative H&E image of synovial explant culture after 3 days of culture. (I) Whole-mount immunoluorescence images showing preservation of endothelial cells after 3 days in culture, marked by CD31 (magenta), and vascular smooth muscle cells marked by MYH11, surrounding CD31 vascular structures. (J) Whole-mount staining showing expression of neurotrophin receptors NGFR, NTRK1, NTRK2, and NTRK3 (green) around vascularature, together with CD31 (magenta) and DAPI (blue). (K) Synovial organoids embedded in Matrigel and treated with NGF, BDNF, or NT3, followed by fixation and staining for a-smooth muscle actin (aSMA) (brown) to visualize mural cells. All 20X images were cropped at similar scale. Scale bar, 150 µm.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Collagen Gel Contraction Assay, Standard Deviation, Preserving, Staining, Expressing

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A) RNAscope images showing transcript expression of pericyte marker RGS5 (yellow) and VSMC marker MYH11 (red) in fibroblasts treated with different neurotrophins, overlaid with DAPI (blue). Scale bar, 150 µm. All images were acquired at 20x magnification and cropped to the same scale.(B) Quantification of total numbers of RGS5+, MYH11+, and RGS5+/MYH11+ double-positive cells following treatment with the indicated neurotrophins.(C) mRNA expression of the pericyte marker RGS5 in fibroblasts transfected with siRNAs targeting NGFR or NTRK1, in the presence or absence of NGF stimulation.(D) mRNA expression of the VSMC marker MYH11 in fibroblasts transfected with siRNAs targeting NTRK2 or NTRK3, in the presence or absence of BDNF or NT3 stimulation, respectively.(E-F) mRNA expression of the mural cell marker CNN1 (calponin) in fibroblasts stimulated with NGF (E) in the presence or absence of a TRKA inhibitor, or stimulated with BDNF (F) in the presence or absence of an NTRK2 inhibitor.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: RNAscope, Expressing, Marker, Transfection

Journal: bioRxiv

Article Title: iPSC modeling of pulmonary arterial hypertension to uncover pathomechanisms and unrecognized modes of action of sotatercept

doi: 10.64898/2026.03.12.711267

Figure Lengend Snippet: (A) Schematic illustration of chemically defined differentiation protocol from iPSCs to iSMCs. (B) Differentiation efficacy determined by flow cytometric analysis of CD140b positive cells. (Data is presented as mean ± SD). (C) Immunofluorescence analysis for SMC markers confirms SMC identity of iSMCs (alpha-smooth muscle actin (Actin) and calponin (CNN1) (both green), myosin heavy chain 11 (MHY11) (red), nuclei (DAPI, blue), scale bar 200 µm. (D) SMC score calculated as the mean expression of Travaglini SMC genes (MSigDB: M41672) in human pulmonary artery smooth muscle cells (GSE144274), human iPSC lines (HipSci resource) and merged expression values of untreated iSMCs from all lines and clones. (E) PCA analysis of untreated iSMCs based on 2000 most variable genes (Log2(TPM+1)) of three independent WT cell lines (dark green dots) and three clones per BMPR2-mutant of, whereas BMPR2 MutExDo (light green dots) and BMPR2 MutKiDo (medium green dots); average of each experimental groups depicted as cross. (F) Quantification of cell numbers relative to the respective vehicle control after 8d treatment. Data is presented as mean ± SD, n= 3 independent biological replicates per column indicated as dots. Exploratory two-way ANOVA with genotype and treatment as factors was followed by Sidak’s multiple-comparisons test to compare genotype differences within Activin A treatment and to compare effects of Activin A, and Activin A in combination with sotatercept, within each genotype. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. (G) Quantification of apoptotic signal measured as caspase3/7 activity and normalization to respective cell number after 5d treatment. Data is presented as mean ± SD, n= 6-9 independent biological replicates per column indicated as dots. Two-way ANOVA with genotype and treatment as factors was followed by Sidak’s multiple-comparisons test to compare genotype differences within Activin A treatment and to compare effects of Activin A and Activin A in combination with sotatercept within each genotype. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: On day 12, for quantification of HPAH phenotypic intracellular or transmembrane markers, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, cat. no.158127) for 10 min, ice-cold 90% methanol (JT Baker, cat. no. 8045) for 15 min, permeabilized with 1% tritonX-100 (Sigma-Aldrich, cat. no. 93443) PBS pH7.4 (1X) (Gibco, cat. no. 70011-036) supplemented with 1 % BSA (Sigma-Aldrich, cat. no. A9418) for 20 min and stained with the primary antibody CNN1 (Santa Cruz Biotechnology, cat. no. sc-53136, RRID:AB_793529, mouse, 1:50) or ITGA2 (Abcam, cat. no. ab181548, RRID:AB_2847852, rabbit, 1:100) in PBS pH7.4 (1X) (Gibco, cat. no. 70011-036) supplemented with 1 % BSA (Sigma-Aldrich, cat. no. A9418) and 1% tritonX-100 (Sigma-Aldrich, cat. no. 93443) over night at 4 °C.

Techniques: Immunofluorescence, Expressing, Clone Assay, Mutagenesis, Control, Activity Assay

Journal: bioRxiv

Article Title: iPSC modeling of pulmonary arterial hypertension to uncover pathomechanisms and unrecognized modes of action of sotatercept

doi: 10.64898/2026.03.12.711267

Figure Lengend Snippet: ( A ) Functional contraction assay with iSMCs derived from representative WT cell line or BMPR2 Mut clones. Double arrow indicates well diameter as starting position for contraction. ( B ) Contraction was determined as the area of the gel disc after 72 h relative to the initial area of the iSMC-gel-discs. Data is presented as mean ± SD, n= 3 independent biological replicates per column indicated as dots. Exploratory two-way ANOVA with genotype and treatment as factors was followed by Sidak’s multiple-comparisons test to compare genotype differences within Activin A treatment and to compare effects of Activin A and Activin A in combination with sotatercept within each genotype. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. ( C ) Quantification of the ratio of phosphorylated myosin light chain (pMLC) to total MLC by automated capillary-based western blot analysis. Data is presented as mean ± SD, n= 3 independent biological replicates per column indicated as dots. Exploratory two-way ANOVA with Sidak’s multiple-comparisons was used to compare genotype differences within Activin A or Activin A in combination with sotatercept, followed by two-tailed unpaired Student’s t-tests for treatment comparisons within each genotype. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. ( D, F ) Immunofluorescence staining of components of the contractile apparatus in iSMCs of one iPSC line / clone representative for the respective genotype (BMPR2 WT , BMPR2 MutExDo , BMPR2 MutKiDo ). Actin and calponin (CNN1) (green), nuclei DAPI (blue), scale bar 200 µm. ( E ) Quantification of actin from immunofluorescence images expressed as mean fluorescence intensity (MFI) normalized to cell number (DAPI). Data is presented as mean ± SD, n= 6 independent biological replicates per column indicated as dots. Two-way ANOVA with genotype and treatment as factors was followed by Sidak’s multiple-comparisons test to compare genotype differences within Activin A treatment and to compare effects of Activin A and Activin A in combination with sotatercept within each genotype. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. ( G ) Quantification of CNN1 from flow cytometry analysis (MFI). Data is presented as mean ± SD, n= 3 independent biological replicates per column indicated as dots. Exploratory two-way ANOVA with genotype and treatment as factors was followed by Sidak’s multiple-comparisons test to compare genotype differences within Activin A treatment and to compare effects of Activin A and Activin A in combination with sotatercept within each genotype. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. ( H ) Heatmaps showing differentially expressed genes with pro-contractile function in Activin A vs. Activin A + sotatercept treated iSMCs each condition normalized to the untreated control. For contraction gene set scoring (MSigDB: M1429), bulk RNA-seq expression values were analysed using a gene-by-sample FPKM matrix (mean FPKM of three independent biological replicates per group, Activin A and Activin A + sotatercept). Stars (*) highlight mentioned genes in the respective text.

Article Snippet: On day 12, for quantification of HPAH phenotypic intracellular or transmembrane markers, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, cat. no.158127) for 10 min, ice-cold 90% methanol (JT Baker, cat. no. 8045) for 15 min, permeabilized with 1% tritonX-100 (Sigma-Aldrich, cat. no. 93443) PBS pH7.4 (1X) (Gibco, cat. no. 70011-036) supplemented with 1 % BSA (Sigma-Aldrich, cat. no. A9418) for 20 min and stained with the primary antibody CNN1 (Santa Cruz Biotechnology, cat. no. sc-53136, RRID:AB_793529, mouse, 1:50) or ITGA2 (Abcam, cat. no. ab181548, RRID:AB_2847852, rabbit, 1:100) in PBS pH7.4 (1X) (Gibco, cat. no. 70011-036) supplemented with 1 % BSA (Sigma-Aldrich, cat. no. A9418) and 1% tritonX-100 (Sigma-Aldrich, cat. no. 93443) over night at 4 °C.

Techniques: Functional Assay, Contraction Assay, Derivative Assay, Clone Assay, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Control, RNA Sequencing, Expressing

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Increased HSD11β1 Expression in Human Leiomyomatous Uteri: Implication for Enhanced Glucocorticoid Signaling

doi: 10.1210/clinem/dgaf255

Figure Lengend Snippet: FKBP51 and dexamethasone induce a switch in leiomyoma cells from a smooth muscle to myofibroblast phenotype. (A) RNA-sequencing analysis of cultured leiomyoma cells (n = 4) shows lower expression levels of extracellular matrix genes including LAMA2 , LAMB1 , and FN1 , and higher expression levels of smooth muscle expressed genes CNN1 , MYH9 , MYH10, and ACTA2 following dexamethasone (DEX) treatment in FKBP5 siRNA (FKsi) vs control siRNA (Csi) transfected cells. (B) Confirmation of significantly lower expression levels of LAMA2 and FN1 , and higher expression levels of CNN1 in FKsi-Dex vs Csi-Dex by qPCR analysis. Bars represent mean ± standard error of the mean; n = 4. RNA sequencing raw data available in the NCBI GEO repository, accession number GSE292403 .

Article Snippet: The full name and Taq-Man Probe Assay IDs for each target gene include: FK506-binding protein 5 ( FKBP5 )- Hs01561006_m1; Hydroxysteroid 11-β dehydrogenase 1 ( HSD11B1) - Hs01547870_m1; IL-1 receptor type 1 ( IL1R1 )- Hs00991002_m1; TSC22 domain family member 3 ( TSC22D3 )- Hs00608272_m1; alcohol dehydrogenase 1B, β polypeptide ( ADH1B )- Hs00605175_m1; Gremlin 1, DAN family BMP antagonist ( GREM1 )- Hs00171951_m1; IGF-binding protein 5 ( IGFBP5 )- Hs00181213_m1; adhesion molecule with Ig-like domain 2 ( AMIGO2 )- Hs05001325_s1; laminin subunit alpha 2 ( LAMA2 )- Hs00166308_m1; fibronectin 1 ( FN1 )- Hs00365052_m1; calponin 1 ( CNN1 )- Hs00959434_m1; actin β ( ACTB )- Hs99999903_m1; and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH )- Hs99999905_m1.

Techniques: RNA Sequencing, Cell Culture, Expressing, Control, Transfection