Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: GPSM3 transcript abundance in whole blood from individuals homozygous for the minor allele of the rs204989/rs204991 haploblock (the “m/m” genotype) is significantly lower than in whole blood from individuals homozygous for the major allele of the haploblock (the “M/M” genotype). ( A ) Schematic representation of identified polymorphisms with respect to the predicted transcription start site of the human GPSM3 gene. ( B ) Scatter plot representation of GPSM3 mRNA abundance, measured in whole blood by qRT-PCR, as normalized to the average level measured in M/M individuals. Individual results for homozygous major allele samples (M/M; n = 53) are depicted by solid circles, and homozygous minor allele samples (m/m; n=11) are depicted by open circles. An average decrease (Δμ) in GPSM3 transcript abundance of 24.1% (t = 3.80, df = 62, p = 0.0003) was observed in homozygous minor allele samples. Error bars represent S.E.M. ( C ) Descriptive statistics of the three cohorts of volunteers genotyped in this study: Minor allele frequencies were 23% for rs204989 and rs204991 in the rheumatoid arthritis samples versus 18% in the disease-free controls (Fisher's exact test; p = 0.4839). Risk/minor allele frequency for rs2812378 was 30.0% in the rheumatoid arthris samples versus 25.0% in the disease-free controls (p = 0.5267). Risk/minor allele frequency for the HLA gene region biallelic SNP rs6457620 was 35.0% in the rheumatoid arthritis samples and 45.0% in the disease-free matched controls (p = 0.1938).

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Quantitative RT-PCR

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Linkage of the GPSM3 SNP haploblock and the known RA risk allele in the HLA gene region, rs6457620 [C>G], is more pronounced in RA samples than controls, but this linkage does not result in a rs6457620 genotype-dependent effect on GPSM3 transcript abundance in whole blood. ( A ) Descriptive statistics of all 200 volunteers, including the RA (n = 50), disease-free matched control (n = 50), and young healthy control volunteers recruited for the current study. These data are stratified by GPSM3 SNP haplotype, showing that the rs6457620 [G] risk allele frequency amongst homozygous ancestral GPSM3 haploblock individuals (M/M) is 41.3%, heterozygous individuals (M/m) is 56.3%, and homozygous minor GPSM3 haploblock (m/m) is 60.7%. These data are significantly different from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0066). ( B ) Analyses of GPSM3 SNP haploblock linkage within RA samples stratified by rs6457620 genotype, again exhibiting statistically significant difference from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0123). ( C ) Within the disease-free matched control samples, GPSM3 SNP minor allele (m) frequency, as stratified by rs6457620 genotype, is not significantly different from chance distribution as determined by Fisher-Freeman-Halton exact test (p = 0.2739). ( D ) Graphical representation of minor allele frequencies (MAFs; of European [‘eu’] population), physical distance (in basepairs), and linkage disequilibrium (LD) value (as quantified by heatmap) between the three linked SNPs of the GPSM3 SNP haploblock (rs204989, rs204990, rs204991) and the HLA gene region SNP rs6547620, as obtained using the NIEHS SNPinfo webserver ( http://snpinfo.niehs.nih.gov/ ) from NCBI dbSNP data (ref. ). ( E ) Despite modest linkage between GPSM3 SNP minor alleles (m) and the rs6457620 risk allele (G), there is no effect of the genotype at HLA gene region SNP rs6457620 on GPSM3 transcript abundance. Significance determined by one-way ANOVA with Bonferroni post-hoc .

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Control

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Functional consequences of SNPs rs204989, rs204990, rs204991, and rs3096688 on GPSM3 promoter activity. ( A ) Functional results upon subcloning a 3.5 kb-region immediately 5′ to the GPSM3 transcription start site from M/M and m/m genotype volunteers, leading to wild type (pGL3-M) and “minor”/polymorphic (pGL3-m) promoter-driven expression of firefly luciferase in HEK293T cells. Luciferase activity is reported as a ratio of GPSM3 promoter-dependent firefly luciferase activity (FLuc) to control Renilla luciferase activity (pRL-TK control vector) and normalized to the average level measured from the wild type pGL3-M vector (set to 1.00; dotted line). Introduction of independent minor alleles (denoted “+###”) into the wild type pGL3-M vector is seen to differentially affect resultant firefly luciferase activity. ( B ) Restoration of the major allele at rs204989 in the pGL3-m vector ( i.e. , “pGL-m-989”) restores wild type GPSM3 promoter activity. All data are compiled from three independent experiments. Error measure is S.E.M. Significance was determined using one-way ANOVA after controlling for multiple comparisons with Dunnett's test using pGL3-M as the defined control.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Functional Assay, Activity Assay, Subcloning, Expressing, Luciferase, Control, Plasmid Preparation

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Putative transcription factor binding sites within the human GPSM3 promoter potentially disrupted by the minor allele of SNP rs204989 (arrows), as predicted by the JASPAR database: ( A ) Fos/Jun (AP1) heterodimer; (B) C/EBP-β.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Binding Assay

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Lentiviral-mediated shRNA knockdown of endogenous GPSM3 expression in the human monocytic THP-1 cell line disrupts migration toward MCP-1 ( A ) Western blot showing whole lysate (40 μg) immunoreactivity with mouse monoclonal anti-GPSM3 antibody 35.5.1 (ref. ) and with anti-β-actin as a loading control. ( B ) Real-time Transwell migration analysis of indicated calcein-AM stained THP-1 cell lines as shown by change in the ratio of MCP-1-induced to vehicle-induced relative fluorescence units (RFU) over a 30 minute timeframe. ( C ) qRT-PCR data from indicated stable knockdown or scrambled control THP-1 cell line total RNA preparations. There was no significant difference in CCR2 mRNA abundance in the THP-1 cell lines as determined by one-way ANOVA (F = 2.40; p = 0.119). ( D ) Regression analysis-derived rate of THP-1 cell line transmigration over initial migratory period; differences between each cell line was calculated by one-way ANOVA. ( E ) Regression analysis-derived maximum THP-1 cell transmigration over 30 minute timecourese; differences between each cell line were calculated by one-way ANOVA. All data are reported as means with error bars representing S.E.M.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: shRNA, Knockdown, Expressing, Migration, Western Blot, Control, Staining, Fluorescence, Quantitative RT-PCR, Derivative Assay, Transmigration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 binds to CD44 which also interacts with IL-13Rα2. a Western blot analysis of CHI3L1, CD44 and IL-13Rα2 protein expression in various gastric cancer cell lines. b - c Lysates from AGS and MGC803 cells were immunoprecipitated (IP) with control IgG and anti-CD44 or anti-CHI3L1 antibody, and then immunoblotted as indicated. Five percent of total cell lysates were used for the input. d Co-localization of CD44 (green) and CHI3L1 (red) in AGS (upper panel), MGC803 (middle panel) and GC tissues from patient #1 (lower panel) by immunofluorescent confocal microscopy (Magnification: 630×). Scale bars represent 10 μm. e - f Lysates from AGS and MGC803 cells were immunoprecipitated (IP) with IgG and anti-CD44 or anti-IL-13Rα2 antibody, and then immunoblotted as indicated. Five percent of total cell lysates were used for the input. g Co-localization of CD44 (green) and IL-13Rα2 (red) in AGS (upper panel), MGC803 (middle panel) and GC tissues from patient #2 (lower panel) by immunofluorescent confocal microscopy (Magnification: 630×). Scale bars represent 10 μm

Article Snippet: RhCD44s extracellular domain (ECD) (Cat. #12211-H08H-50) and recombinant human IL-13Rα2 ECD (Cat. #10350-H08H-20) was obtained from Sino Biological (Beijing, China).

Techniques: Western Blot, Expressing, Immunoprecipitation, Confocal Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 triggers Erk and Akt signaling through CD44. a and b Western blot analysis of Erk and Akt activation in the AGS and MGC803 cells which were pre-exposed with the control antibody or functional CD44 blocking antibody and treated with rhCHI3L1 (500 ng/ml) for the indicated times. c and d Western blot analysis of CD44 and IL-13Rα2 knockdown efficacy in AGS ( c ) and MGC803 ( d ) transfected with scramble, CD44 or IL-13Rα2 shRNA. e and f Westen blot evaluation of the activation of Erk and Akt in AGS and MGC803 cells which stably expressing scrambled, CD44 or IL-13Rα2 shRNA and were cultured in the presence or absence of rhCHI3L1 (500 ng/ml). g Westen blot analysis of the activation of Erk and Akt in bone marrow-derived macrophages (BMDM) from WT and CD44 −/− mice which were incubated with the recombinant mouse CHI3L1 (500 ng/ml) for the noted periods of time

Article Snippet: RhCD44s extracellular domain (ECD) (Cat. #12211-H08H-50) and recombinant human IL-13Rα2 ECD (Cat. #10350-H08H-20) was obtained from Sino Biological (Beijing, China).

Techniques: Western Blot, Activation Assay, Functional Assay, Blocking Assay, Transfection, shRNA, Stable Transfection, Expressing, Cell Culture, Derivative Assay, Incubation, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CD44v3 physically interacts with CHI3L1 and IL-13Rα2. a Illustration of CD44 gene and alternative spliced variants (e.g., CD44s, CD44v3, v6 and v9 isoforms) which contain external, transmembrane (TM) and intracellular domain. b The structure of CD44v3 transmembrane protein, which contains the hyaluronic acid (HA) binding sites at the external N-terminal region, a heparin sulfate (HS) assembly site in v3 domain, and the signaling regulator binding sites at the cytoplasmic region. CD44v3 domain amino acid sequences and v3 peptide used in the current study (in red) were listed. c - e Binding of CD44v3 extracellular domain (ECD) ( c ) or CD44v3 peptide ( d and e ) to rhCHI3L1 or rhIL-13Rα2 ECD. The binding affinity was evaluated by the absorbance at 450 nm in a direct ELISA. Results shown are representative of a minimum of three independent experiments. The values represent the mean ± SEM in triplicate; *p < 0.05. f and g Measurement of the binding affinity of CD44v3 peptide to rhCHI3L1 ( f ) or rhIL-13Rα2 ECD ( g ) by biolayer interferometry (BLI). Various concentrations of CD44v3 peptide were shown. All experiments were performed in triplicate. h Binding of CD44v6 peptide to rhCHI3L1 or rhIL-13Rα2 ECD evaluated by a direct ELISA as describe above. i - j Measurement of the binding affinity of CD44s ECD to rhCHI3L1 ( i ) or rhIL-13Rα2 ECD ( j ) by biolayer interferometry (BLI). CD44s ECD was immobilized and CHI3L1 (500 μM) or IL-13Rα2 (500 μM) was in the mobile phase

Article Snippet: RhCD44s extracellular domain (ECD) (Cat. #12211-H08H-50) and recombinant human IL-13Rα2 ECD (Cat. #10350-H08H-20) was obtained from Sino Biological (Beijing, China).

Techniques: Binding Assay, Direct ELISA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 mediated tumorigenesis through CD44 in vivo. a A representative image of tumor growth in nude mice subcutaneously inoculated with lenti-shCHI3L1- or lenti-shControl-transfected MGC803 cells. b The growth curve of subcutaneous tumor from MGC803 cells infected with lenti-shCHI3L1 or lenti-shControl in nude mice ( n = 10 animals per group). c Comparison of tumor weight from two groups at the end of the experiment. d Evaluation of cell proliferative activity by Ki-67 staining in subcutaneous xenografts from MGC803 cells infected with lenti-shControl or lenti-shCHI3L1. e and f H&E staining of the representative SGC7901 cells lung metastatic lesions in nude mice ( e ). The total area of invasive lesions on the lung slice section represents the invasive tumor volume in the lungs ( f ). Sizing bar indicates 100 μm. g Representative photograph of lungs from WT and CD44 −/− mice 2 weeks after challenge with B16-F10 melanoma cells. Comparison of the number of pleural melanoma colonies in lungs from tumor cells challenged WT and CD44 −/− mice ( n = 8 animals per group). h and i The levels of total ( h ) and active ( i ) TGF-β1 in broncho-alveolar lavage (BAL) fluids from WT mice and CD44 −/− mice. j Schematic representation of the CHI3L1/CD44-IL-13Rα2 signaling pathway in promoting GC cell metastasis. Data are presented as mean ± SEM, * p < 0.05 (Student’s t test)

Article Snippet: RhCD44s extracellular domain (ECD) (Cat. #12211-H08H-50) and recombinant human IL-13Rα2 ECD (Cat. #10350-H08H-20) was obtained from Sino Biological (Beijing, China).

Techniques: In Vivo, Transfection, Infection, Activity Assay, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic guanosine monophosphate-protein kinase G signaling attenuates aortic valve calcification through ULK1-mediated autophagy

doi: 10.1038/s41392-026-02624-5

Figure Lengend Snippet: PKGI directly phosphorylates ULK1 at Ser556 to activate autophagy and inhibit the osteogenic differentiation of hVICs. a Overview of significantly up- and downregulated phosphosites across the control medium (NC), osteogenic medium (OM), OM plus vericiguat (VER), and OM plus vericiguat with PKGI inhibitor (PKGi) comparisons in hVICs via phosphoproteomic analysis (Student’s t -test, P < 0.05, fold change > 1.5). b Venn diagram showing the phosphosites oppositely regulated by vericiguat treatment and vericiguat plus PKGI inhibition. c , d Bioinformatics enrichment analyses (KEGG and ClueGO) highlighting the significant enrichment of differentially phosphorylated proteins in autophagy-related pathways. e LC‒MS/MS analysis identifying ULK1 as the top-ranked interacting protein of PKGI in FLAG-PKGI pulldown samples compared with IgG controls. f Protein–protein docking analysis indicating a strong and stable interaction between ULK1 and PKGI. g Scatter plot showed three phosphorylation sites on ULK1 (Ser477, Ser479, and Ser556) that exhibit opposite phosphorylation patterns between the Vericiguat-treated and PKGI-inhibited groups. h , i Co-IP assays confirming the direct interaction between FLAG-tagged PKGI and HA-tagged ULK1 in 293 T cells. j Autoradiography-based ATP-γ-S kinase assay confirming direct phosphorylation of ULK1 by PKGI in vitro. k – n Alizarin Red staining, ALP activity ( k ), and Western blot analysis ( m ) were used to evaluate the functional relevance of the ULK1 phosphorylation sites. hVICs transfected with WT-ULK1 or phosphorylation-deficient mutants (S556A, S477A, and S479A) were treated with 8-Br-cGMP under osteogenic conditions. Mutation at S556A abolished PKGI-mediated suppression of osteogenic marker expression and calcification, whereas mutations at Ser477 and Ser479 had no effect ( n = 3–6 independent experiments; mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test; exact P values are indicated)

Article Snippet: For the ATP-γ-S kinase assay, 5 μM recombinant His-tagged human PRKG1 (His-hPRKG1, Novoprotein, Cat. No. C923) and 5 μM recombinant GST-tagged ULK1 (GST-ULK1, SinoBiological, Cat. No. U01--11G) were incubated separately with 20 μM ATP (MCE, Cat. No. HY-B2176) in 1× kinase reaction buffer (MCE, Cat. No. HY-K0016) at 37 °C for 2 h to facilitate autophosphorylation.

Techniques: Control, Inhibition, Phospho-proteomics, Co-Immunoprecipitation Assay, Autoradiography, Kinase Assay, In Vitro, Staining, Activity Assay, Western Blot, Functional Assay, Transfection, Mutagenesis, Marker, Expressing

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 1. ENN B1 inhibits the viability and expression of functional genes in Leydig cells. A, Leydig cells (TM3 cells) were treated with various doses of ENN B1 (5 μM, 10 μM, 20 μM, 40 μM, 50 μM) for 24 h, and cellular viability was measured. B, Leydig cells (TM3 cells) were treated with ENN B1 (10 μM) for different times (12 h, 24 h, 36 h, 48 h, 60 h), and cellular viability was measured. C, TM3 cells were treated with the indicated doses of ENN B1 for 24 h, and total cellular lysates were subjected to western blotting with the indicated antibodies. GAPDH and tubulin were used as the loading controls. D, TM3 cells were treated with the indicated doses of ENN B1 for 24 h, and the indicated mRNA levels of various genes were measured by qRT–PCR. E, TM3 cells were treated with hCG (50 ng/ml) for 12 h, and then the cells were treated with the indicated doses of ENN B1 for another 24 h. The levels of progesterone in the supernatant were measured by ELISA. F, TM3 cells were treated with the indicated doses of ENN B1 for 24 h, and the levels of testosterone were measured by ELISA. The data are presented as the mean ± SD; *P<0.05; **P<0.01, ***P<0.01.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: Expressing, Functional Assay, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 2. ENN B1 triggers apoptosis in a caspase-dependent manner in Leydig cells. A, TM3 cells were treated with the indicated doses of ENN B1 for 24 h, and cellular apoptosis was measured by Annexin V-FITC staining assays. B, DNA fragmentation was measured. C, Relative caspase-3 activities were measured. D, Total cellular lysates were subjected to the indicated antibodies. E, TM3 cells were treated with various doses of ENN B1 with or without z. After treatment with VAD (5 μM) for 24 h, cellular apoptosis was measured. The data are presented as the mean ± SD; *P<0.05; **P<0.01.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: Staining

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 3. ENN B1 induces apoptosis via upregulation of Bax and downregulation of Bcl-2 in Leydig cells. A, TM3 cells were treated with the indicated doses of ENN B1 for 24 h, and total cellular lysates were subjected to western blot analysis with various antibodies. B, TM3 cells were transfected with the indicated shRNAs for 24 h, and the protein levels of Bax were measured by western blotting. C, TM3 cells were transfected with the indicated shRNAs for 24 h, then cells were treated with or without ENN B1 (20 μM) for another 24 h, and cellular apoptosis was measured. D, The activation of caspase-3 was examined by western blotting. E, TM3 cells were transfected with empty vector or Bcl-2 overexpressing vector (Bcl-2 OV) for 24 h, and the levels of Bcl-2 were measured by western blots. F, TM3 cells were transfected as indicated for 24 h, then cells were treated with or without ENN B1 for another 24 h, and cellular apoptosis was measured. The data are presented as the mean ± SD; **P<0.01.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: Western Blot, Transfection, Activation Assay, Plasmid Preparation

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 4. ENN B1 induces oxidative stress in Leydig cells. A, TM3 cells were treated with the indicated doses of ENN B1 for 24 h, and the levels of ROS and MDA and the relative activities of SOD and CAT were measured. B, TM3 cells were pretreated with or without NAC (10 μM) for 4 h, then treated with or without ENN B1 (20 μM) for another 24 h, and cellular viability was measured. C, Cellular apoptosis was measured. D, the activation of caspase-3 was examined by western blotting. E, mRNA levels of various functional genes were measured by qRT–PCR. F, The protein levels of the indicated functional genes were measured by western blotting. The data are presented as the mean ± SD; *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: Activation Assay, Western Blot, Functional Assay, Quantitative RT-PCR

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 6. ENN B1 represses the STAT3 signaling pathway in Leydig cells. A, TM3 cells were treated with various doses of ENN B1 for 24 h, and the indicated proteins were measured using western blot assays. B, Dual-luciferase activity assays were conducted to verify the effects of ENN B1 on STAT3 signaling. C, TM3 cells were treated with ENN B1 (20 μM) alone or in combination with sodium pervanadate for 24 h, and total cellular lysates were subjected to western blots with the indicated antibodies. D, Cellular viability was measured. E, Cellular apoptosis was measured. F, Cleavage of caspase-3 was assayed by western blotting. G, mRNA levels of various functional genes were measured by RT–PCR. H, Protein levels of various functional genes were measured by western blots. The data are presented as the mean ± SD; *P<0.05; **P<0.01.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: Western Blot, Luciferase, Activity Assay, Functional Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 7. ENN B1 inhibits STAT3 signaling in an SHP-1-dependent manner in Leydig cells. A, TM3 cells were treated with various doses of ENN B1 for 24 h, and the indicated proteins were measured using western blot assays. B, TM3 cells were transfected with the indicated shRNAs for 24 h, and the protein levels of SHP-1 were measured by western blotting. C, TM3 cells were transfected with the indicated shRNAs for 24 h, and then the cells were treated with or without ENN B1 (20 μM) for another 24 h. Total cellular lysates were subjected to western blotting with the indicated antibodies. D, Cellular viability was measured. E, Cellular apoptosis was measured. F, Cleavage of caspase-3 was assayed by western blotting. G, mRNA levels of various functional genes were measured by RT–PCR. H, Protein levels of various functional genes were measured by western blots. The data are presented as the mean ± SD; *P<0.05; **P<0.01.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: Western Blot, Transfection, Functional Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Ecotoxicology and environmental safety

Article Title: Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

doi: 10.1016/j.ecoenv.2024.116116

Figure Lengend Snippet: Fig. 8. The effect of ENN B1 on testicular toxicity in vivo. A, The body weights of mice were recorded during the exposure period and weekly body weights were recorded (n=6 in each group). B, The weight of the testis after the mice were sacrificed. C, Testis/body weight index of four groups. D, Representative histology of the testis from each group. Red arrows indicate deciduous germ cells in seminiferous tubules. E, Serum testosterone levels in different groups. F, Testicular testosterone levels in different groups. G, Serum levels of LH and GnRH. I, LDH activity in testis tissues. J, SDH activity in testis tissues. K, The number of sperm from cauda epididymides. L, Sperm motility was measured. M, testis tissue was subjected to western blots analysis with indicated antibodies. N, Schematic illustration of ENN B1-induced apoptosis and dysfunction in TM3 cells. The data are presented as the mean ± SD; *P<0.05; **P<0.01.

Article Snippet: ENN B1 (Cat No: HY-N3807) was obtained from MedChemExpress (USA) and prepared according to the manufacturer’s instructions. z. VAD.fmk (Cat No: S8102) and NAC (Cat No: S1623) were obtained from Selleck (USA).

Techniques: In Vivo, Activity Assay, Western Blot

Journal: International Journal of Oncology

Article Title: Carcinoembryonic antigen is a sialyl Lewis x/a carrier and an E-selectin ligand in non-small cell lung cancer

doi: 10.3892/ijo.2019.4886

Figure Lengend Snippet: CEA glycoprotein is a functional E-selectin ligand in NSCLC. (A) Adhesion of E-selectin-expressing cells to NSCLC lysates. Lysates of NSCLC tissue, derived from patient 7, were spotted on glass slides, and the adhesion of CHO-E cells was tested using a modified Stamper-Woodruff binding assay test. As E-selectin interactions are calcium-dependent, CHO-E cells were resuspended in calcium buffer. EDTA buffer was used as a control. Cells were pre-incubated with 20 µ g/ml of specific mAbs prior to the adhesion experiment; isotype control or function-blocking anti-E-selectin mAb. (B) Adhesion of E-selectin-expressing cells to CEA immunoprecipitate. CEA was immunoprecipitated from NSCLC tissue derived from patient 7 and spotted on glass slides. Adhesion of CHO-E or CHO-mock cells to CEA IP was tested using a modified Stamper-Woodruff binding assay. CHO-E and CHO-mock cells were resus-pended in calcium buffer, and CHO-E cells resuspended in EDTA buffer were used as a negative control. One-way ANOVA followed by Tukey's post hoc test was used for analysis. ** P<0.01. Representative pictures captured for each condition (magnification, ×100) are represented below the respective graphs. CD62E, E-selectin; CEA, carcinoembryonic antigen; CHO, Chinese hamster ovary cells; CHO-E, E-selectin-expressing CHO; CHO-mock, empty vector-transfected CHO; isotype, isotype control Ab; mAb, monoclonal antibody.

Article Snippet: In certain cases, CHO-E cells were previously incubated with 20 µ g/ml of function-blocking anti-CD62E (clone 68-5H11; cat. no. 555648; BD Biosciences) or isotype control mAb (clone mopc-21; cat. no. 400102; BioLegend, Inc.).

Techniques: Functional Assay, Expressing, Derivative Assay, Modification, Binding Assay, Incubation, Blocking Assay, Immunoprecipitation, Negative Control, Plasmid Preparation, 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: Frontiers in Immunology

Article Title: TIPE2 May Target the Nrf2/HO-1 Pathway to Inhibit M1 Macrophage–Related Neutrophilic Inflammation in Asthma

doi: 10.3389/fimmu.2022.883885

Figure Lengend Snippet: Demographic, functional, and inflammatory characteristics of asthma patients and healthy volunteers.

Article Snippet: The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: TIPE2 May Target the Nrf2/HO-1 Pathway to Inhibit M1 Macrophage–Related Neutrophilic Inflammation in Asthma

doi: 10.3389/fimmu.2022.883885

Figure Lengend Snippet: (A) Induced sputum concentrations of TIPE2 in the asthma inflammatory phenotypes. (B) Sputum cells from NA, PA, and EA patients stained for TIPE2 (red); nuclei were stained with DAPI (blue). NA, neutrophilic asthma; EA, eosinophilic asthma; MA, mixed granulocytic asthma; PA, paucigranulocytic asthma. *P<0.05, **P<0.01.

Article Snippet: The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: TIPE2 May Target the Nrf2/HO-1 Pathway to Inhibit M1 Macrophage–Related Neutrophilic Inflammation in Asthma

doi: 10.3389/fimmu.2022.883885

Figure Lengend Snippet: Correlations between TIPE2 and inflammatory mediators/clinical characteristics. TNFAIP8, tumor necrosis factor-alpha-induced protein–8; TIPE, TNFAIP8-like; IL, interleukin; TNF-α, tumor necrosis factor-α; FeNO, fractional exhaled nitric oxide.

Article Snippet: The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Techniques:

Journal: Frontiers in Immunology

Article Title: TIPE2 May Target the Nrf2/HO-1 Pathway to Inhibit M1 Macrophage–Related Neutrophilic Inflammation in Asthma

doi: 10.3389/fimmu.2022.883885

Figure Lengend Snippet: TIPE2 impeded M1 macrophage differentiation and inhibited the expression and release of related inflammatory cytokines. (A) TIPE2 expression was down-regulated in M1 macrophages. TIPE2 expression levels were detected by RT-qPCR and western blotting analysis. (B) THP-1 monocytes were transduced with lentivirus expressing TIPE2 shRNA or TIPE2 overexpression sequences. TIPE2 expression levels were determined by the same method as above. (C, D) Expression of M1 genes in THP-1 monocytes by RT-qPCR following treatment with PMA (20 ng/ml) for 48 h and LPS (1 µg/ml) for 24 h. Measurements of IL-1β, IL-6, and TNF-α mRNA expression levels in THP-1 monocytes by RT-qPCR following treatment with PMA (20 ng/ml) for 48 h and LPS (1 µg/ml) for 2 h. (E) Measurement of IL-1β, IL-6, and TNF-α secretion in THP-1 monocytes by ELISA following treatment with LPS (1 µg/ml) for 2 h. Statistics were performed on pooled data from at least three independent experiments. Error bars represent the standard deviations of the means. A comparison of two groups was performed using Student’s t-test. Comparison of the means among three groups was performed using one-way ANOVA followed by post-hoc test (LSD or Tamhane’s T2). * P<0.05, ** P<0.01, *** P<0.001. TIPE2, tumor necrosis factor α–induced protein 8–like protein 2; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; shRNA, short hairpin RNA; TIPE2-OE, TIPE2 overexpression; iNOS, inducible nitric oxide synthase; IL, interleukin; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; PMA, phorbol-12-myristate-13-acetate; CTRL, control vector; ANOVA, analysis of variance; LSD, least significant difference.

Article Snippet: The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transduction, shRNA, Over Expression, Enzyme-linked Immunosorbent Assay, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TIPE2 May Target the Nrf2/HO-1 Pathway to Inhibit M1 Macrophage–Related Neutrophilic Inflammation in Asthma

doi: 10.3389/fimmu.2022.883885

Figure Lengend Snippet: TIPE2 promotes Nrf2/HO-1 signaling pathway activation. (A, B) Expression of Nrf2 and HO-1 in THP-1 monocytes by RT-qPCR following treatment with PMA (20 ng/ml) for 48 h and LPS (1 µg/ml) for 24 h. (C, D) Nrf2 and HO-1 protein levels in THP-1 monocytes were detected by western blotting analysis following treatment with PMA (20 ng/ml) for 48 h and LPS (1 µg/ml) for 24 h. (E) Expression of nuclear and cytoplasmic Nrf2 in M1 macrophages was detected by western blotting analysis. (F) Expression of Nrf2 in M1 macrophages was detected by immunocytochemistry analysis. Statistics were performed on pooled data from at least three independent experiments. Error bars represent the standard deviations of the means. A comparison of two groups was performed using Student’s t-test. Comparison of the means among three groups was performed using one-way ANOVA followed by post-hoc test (LSD or Tamhane’s T2). * P<0.05, ** P<0.01. TIPE2, tumor necrosis factor α–induced protein 8–like protein 2; Nrf2, nuclear factor erythroid 2–related 2; HO-1, heme oxygenase-1; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; shRNA, short hairpin RNA; TIPE2-OE, TIPE2 overexpression; LPS, lipopolysaccharide; PMA, phorbol-12-myristate-13-acetate; CTRL, control vector; ANOVA, analysis of variance; LSD, least significant difference.

Article Snippet: The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Over Expression, Control, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TIPE2 May Target the Nrf2/HO-1 Pathway to Inhibit M1 Macrophage–Related Neutrophilic Inflammation in Asthma

doi: 10.3389/fimmu.2022.883885

Figure Lengend Snippet: Nrf2 inhibitor weakened the anti-inflammatory role of TIPE2 in M1 macrophage polarization. (A) Expression of Nrf2 and HO-1 in M1 macrophages by RT-qPCR following treatment with the Nrf2 inhibitor ML385 (5 nmol) for 48 h. (B) Measurement of Nrf2 expression level in M1 macrophages by immunocytochemistry analysis following treatment with ML385 (5 nmol) for 48 h. (C) The protein levels of Nrf2 and HO-1 in M1 macrophages were detected by western blotting analysis following treatment with ML385 (5 nmol) for 48 h. (D) Measurement of CD11c, iNOS, IL-1β, IL-6, and TNF-α expression levels in M1 macrophages by RT-qPCR following treatment with ML385 (5 nmol) for 48 h. Statistics were performed on pooled data from at least three independent experiments. Error bars represent the standard deviations of the means. A comparison of two groups was performed using Student’s t-test. Comparison of the means among three groups was performed using one-way ANOVA followed by post-hoc test (LSD or Tamhane’s T2). * P<0.05, ** P<0.01. TIPE2, tumor necrosis factor α–induced protein 8–like protein 2; Nrf2, nuclear factor erythroid 2–related 2; HO-1, heme oxygenase–1; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; shRNA, short hairpin RNA; TIPE2-OE, TIPE2 overexpression; iNOS, inducible nitric oxide synthase; IL, interleukin; TNF-α, tumor necrosis factor–α; CTRL, control vector; ANOVA, analysis of variance; LSD, least significant difference.

Article Snippet: The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Immunocytochemistry, Western Blot, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Over Expression, Control, Plasmid Preparation

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: The following primary antibodies were used: Vinculin ((Sigma-Aldrich,cat. no. V9131, RRID:AB_477629, mouse, 1:125.000), total myosin light chain (Cell Signaling Technology, cat. no. 8505, RRID:AB_2728760, rabbit, 1:20), phosphorylated myosin light chain (Cell Signaling Technology, cat. no. 3671, RRID:AB_330248, rabbit, 1:50).

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: The following primary antibodies were used: Vinculin ((Sigma-Aldrich,cat. no. V9131, RRID:AB_477629, mouse, 1:125.000), total myosin light chain (Cell Signaling Technology, cat. no. 8505, RRID:AB_2728760, rabbit, 1:20), phosphorylated myosin light chain (Cell Signaling Technology, cat. no. 3671, RRID:AB_330248, rabbit, 1:50).

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