Journal: bioRxiv

Article Title: Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

doi: 10.1101/2025.09.22.677845

Figure Lengend Snippet: a . Western blot showing time course of TGFβ1 or Latency associated peptide (LAP) (TGFβ) expression in the heart at embryonic day 16.5 (E16.5), postnatal day 1 (P1), P7, P21, and 12 weeks of age. Gapdh was used to normalize protein loading. b . Western blot for TGFβ1 and Cre from protein extracts of neonatal cardiomyocytes from Tgfb1/2/3 fl/fl-αMHC-Cre mice transduced with Ad-GFP or Ad-Cre 48 hrs prior in culture. c . mRNA expression of Tgfb1 , Tgfb2 , and Tgfb3 from neonatal cardiomyocytes transduced with Ad-GFP (adenovirus expressing green fluorescent protein) or Ad-Cre (adenovirus expressing Cre recombinase). n=2-5 per treatment group, *p<0.05. d . Schematic of the generation of Tgfb1/2/3 fl/fl-αMHC-Cre mice whereby Tgfb1 fl/fl , Tgfb2 fl/fl , and Tgfb3 fl/fl mice were crossed to the αMHC-Cre transgene. e . Kaplan-Meier curve depicting survival of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice over time in weeks. f . Heart-weight to body-weight (HW/BW) ratio of Tgfb1/2/3 fl/fl , Tgfb1/2/3 fl/fl-αMHC-Cre , and αMHC-Cre mice at 6 weeks of age. n=4-19 mice in each group, *p<0.05. g . Fractional shortening (FS%) at 6 weeks of age of the same groups of mice as in f. n=6-17 mice per group. *p<0.05. h . Left ventricular internal diameter in diastole (LVID;d) at 6 weeks of age in the same groups of mice as in f. n=6-17 mice per group. *p<0.05. i . Representative histological sections from hearts stained with Sirius Red to assess collagen deposition and wheat germ agglutinin-FITC (WGA, green) to assess cardiomyocyte cross sectional area (CSA) of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice at 6 weeks of age. Scale bar Sirius Red: 2 mm; Scale bar WGA: 100 µm. j . Quantification of average cardiomyocyte CSA from WGA staining as shown in i. A minimum of 250 myocytes were measured from each heart, across 3 hearts (points on the graph). *p<0.05. k . Hydroxyproline quantification of fibrosis from the left ventricle of hearts at 6 weeks of age as shown in j. n=8-9 mice per group. *p<0.05. l . Flow cytometry quantification of MEFSK4+ fibroblasts at 6 weeks of age in the hearts of mice shown in j. n=9-10 mice per group. *p<0.05. c,j,l. Data are presented as mean +/-SEM. Statistical analysis was performed using Student’s T-tests. f-h. Statistical analysis was performed using One Way ANOVAs with Tukey’s multiple comparisons test. k. In the case of non-normally distributed data, Mann Whitney tests were performed. Data are presented as mean +/-SEM.

Article Snippet: Fluorophore-conjugated antibodies against MEFSK4-allophycocyanin (APC) (Miltenyi Biotec, Waltham, MA, USA; 130-120-802;1:50), CD45-fluorescein isothiocyanate (FITC) (BD Biosciences, Franklin Lakes, NJ, USA; 563890;1:200), and CD31-brilliant violet 421 (BioLegend, San Diego, CA, USA; 102423;1:200) were added to samples and incubated on ice for 30 minutes.

Techniques: Western Blot, Expressing, Transduction, Staining, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

doi: 10.1101/2025.09.22.677845

Figure Lengend Snippet: a . Images of hearts at P1 and P7 from the two genotypes of mice demonstrating early dysmorphia. Scale bar: 1 mm (equal aspect ratio in both pictures). b . Heart-weight to body weight ratio (HW/BW) at P7 in the three genotypes of mice shown. n=8-14 mice per group. *p<0.05. c . Hydroxyproline quantification of fibrosis from left ventricles of mice at P7 in the three genotypes of mice shown. n=4-6 mice per group. d . Flow cytometric quantification of MEFSK4+ fibroblasts from left ventricles of mice at P7 in the two genotypes of mice shown. n=8 mice per group. e . Atomic force microscopy quantification of cardiac stiffness at P7 in hearts of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice. A total of 64 force curves were generated from histological sections in minimally 10 different areas of the LV free wall (minimally 640 force curves analyzed/heart). Each datum point depicted represents the average of the force curves generated for a given heart. n=5 mice per group. *p<0.05. f . Western blot assessment of extracellular matrix proteins in the hearts of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice at P7. GAPDH was used as a processing and loading control. g . Representative immunostaining of cardiac histological sections for WT1 (green) merged with laminin (red) to assess epicardial derived cells migrating into the myocardium at E18.5 for the two genotypes shown. Scale bars: 100 µm. h . mRNA expression of genes involved in epithelial-mesenchymal transition (EMT), smooth muscle cell (SMC) maturation, and extracellular matrix (ECM) expression in E14.5 hearts of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice. Genes were normalized to 18S ribosomal RNA expression. n=4-6 mice per group. b,c. Data presented as mean +/-SEM. Kruskal Wallis tests were used with Dunn’s multiple comparisons test for statistical analysis. d,e,h. Data presented as mean +/-SEM. Student’s T-test used for statistical analysis.

Article Snippet: Fluorophore-conjugated antibodies against MEFSK4-allophycocyanin (APC) (Miltenyi Biotec, Waltham, MA, USA; 130-120-802;1:50), CD45-fluorescein isothiocyanate (FITC) (BD Biosciences, Franklin Lakes, NJ, USA; 563890;1:200), and CD31-brilliant violet 421 (BioLegend, San Diego, CA, USA; 102423;1:200) were added to samples and incubated on ice for 30 minutes.

Techniques: Microscopy, Generated, Western Blot, Control, Immunostaining, Derivative Assay, Expressing, RNA Expression

Journal: Molecules

Article Title: Enhancing Aseptic Inflammation Resolution with 1-(2-Ethoxyethyl)-4-(pent-1-yn-1-yl)piperidin-4-yl Propionate: A Novel β-Cyclodextrin Complex as a Therapeutic Agent

doi: 10.3390/molecules29215135

Figure Lengend Snippet: The proportion of CD4 + and CD4 + CD25 + lymphocytes was analyzed in animals with aseptic inflammation treated with EPPPβCD (immunophenotyping of spleen cells from the Control, SI, Me/SI, and Me/SI/ EPPPβCD groups was conducted on the 7th and 14th days. The results are categorized into the following: ( A )—%CD4 + lymphocytes; ( B )—%CD4 + CD25 + lymphocytes. Significant differences between the groups were determined using the Student’s t -test, with significance thresholds set at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After lysis, the cells were washed twice in 2 mL and, subsequently, the cells were resuspended in 100 μL of chilled physiological solution, after which cell surface markers were stained using monoclonal antibodies to CD4 (0.25 μg/sample, clone OX35, conjugated with the fluorophore PerCP-eFluor™ 710 (eBioscience™) and monoclonal antibodies to CD25 (0.125 μg/sample, clone OX39, APC fluorophore, eBioscience™) for 30 min in the dark when cooled to 4 °C.

Techniques: Control

Journal: Molecules

Article Title: Enhancing Aseptic Inflammation Resolution with 1-(2-Ethoxyethyl)-4-(pent-1-yn-1-yl)piperidin-4-yl Propionate: A Novel β-Cyclodextrin Complex as a Therapeutic Agent

doi: 10.3390/molecules29215135

Figure Lengend Snippet: The proportion of CD4 + and CD4 + CD25 + lymphocytes was analyzed in animals with aseptic inflammation treated with EPPPβCD (immunophenotyping of spleen cells from the Control, SI, Me/SI, and Me/SI/ EPPPβCD groups was conducted on the 7th and 14th days. The results are categorized into the following: ( A )—%CD4 + lymphocytes; ( B )—%CD4 + CD25 + lymphocytes. Significant differences between the groups were determined using the Student’s t -test, with significance thresholds set at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The experimental protocol involved the use of the following reagents: Phosphate Buffer Saline (PBS, Sigma-Aldrich, Darmstadt, Germany), 0.9% saline solution (Kelun-Kazpharm LLP, Almaty, Kazakhstan), nylon filters with a mesh size of 70 µm (Fisherbrand™, Leicestershire, UK), High-Yield Lyse reagent for erythrocyte lysis (Invitrogen™, Alcobendas, Spain), CytoFix Fixation Buffer for cell fixation (BD Biosciences, San Jose, CA, USA), monoclonal antibodies against CD4 (0.25 µg/sample, clone OX35, PerCP-eFluor™ 710 fluorophore, eBioscience™, Torrance, CA, USA), and monoclonal antibodies against CD25 (0.125 µg/sample, clone OX39, APC fluorophore, eBioscience™, USA).

Techniques: Control