primary human cardiac fibroblasts hcf  (PromoCell)

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: a. Binder scaffolds used for mitochondria delivery. b. Schematic of full-length antibody conjugation to SNAP-tag-displaying mitochondria. c. HEK293T cells expressing a SNAP-tag at the outer membrane of mitochondria. The SNAP-tag is stained by anti-SNAP antibodies (magenta). Mitochondria are stained by anti-MT-CO1 antibodies (green). The SNAP-tag display on the mitochondrial surface was validated in at least three independent experiments. d. Immunostaining of isolated mitochondria displaying benzylguanine (BG)-conjugated anti-CD31 antibodies bound to the mitochondria outer membrane displayed SNAP-tag (bottom). Isolated mitochondria without BG-anti-CD31 antibodies are shown at the top. Mitochondria were detected by anti-SNAP-tag antibodies (magenta). Mitochondria-displayed anti-CD31 antibodies were detected with anti-mouse IgG conjugated with Alexa fluor-647 conjugated antibodies (cyan). e. Immunostaining of isolated mitochondria displaying BG-conjugated isotype control IgG1 bound to the mitochondria outer membrane-displayed SNAP-tag (bottom). Isolated mitochondria without BG-IgG1 (top). Mitochondria were detected by anti-SNAP-tag antibodies (magenta). Mitochondria-displayed IgG1 were detected with anti-mouseIgG conjugated with Alexa fluor-647 conjugated antibodies (cyan). f. Colocalization of SNAP-tag with either BG-anti-CD31 (left) or BG-IgG1 (right) in (d) and (e), respectively. n = 5, P < 0.0001, two-sided paired t test. g. Western blotting on isolated mitochondria displaying a SNAP-tag fused to a full-length antibody. Isolated mitochondria were incubated at different concentrations with either BG-conjugated anti-CD31 antibodies (BG-anti-CD31) or IgG1 (BG-IgG1). For controls, BG-free antibodies were incubated with isolated mitochondria displaying a SNAP-tag (SNAP-OMP25). Loading control, anti-TOMM20 antibody. A SNAP-tag interacting with the BG-conjugated antibody was detected with anti-SNAP-tag antibodies. Shifting in weight indicates SNAP-tag interaction with the light or heavy chain (LC/HC) of the BG-conjugated antibody. n × SNAP-OMP25, multiple SNAP-tag bound to LC or HC. For gel source data, see Supplementary Fig. . The experiment was repeated two times. h. Endothelial cells targeted by donor mitochondria displaying either IgG1 or anti-CD31 antibodies two hours after transplantation. Endothelial cells are stained by Phalloidin dye (green). Donor mitochondria displaying antibodies are stained by anti-SNAP antibodies (magenta). Cells are outlined with grey dashed lines. i. Quantification of the efficacy of the delivery of antibody-displaying mitochondria two hours after transplantation. n = 5, top: P = 0.1051 (10 nM), 0.0016 (100 nM), 0.0008 (500 nM) and bottom P = 0.7501 (10 nM), 0.0079 (100 nM), 0.0013 (500 nM), two-sided Welch’s t test and Mann-Whitney test (bottom 100 nM). Effect of avidity increase on mitochondria targeting efficiency (percentage) for anti-CD31: P = 0.0133 and for control IgG: P = 0.1432, Welch’s ANOVA test. Effect of avidity increase on mitochondria targeting efficiency (ratio) for control IgG: P = 0.0057, Welch’s ANOVA test. j. Schematic of mitochondria delivery displaying either BG-anti-CD31 or BG-IgG1 into primary endothelial cells (shown in magenta, CD31-positive) and cardiac fibroblasts (shown in brown, CD31-negative). k. Endothelial cells and cardiac fibroblasts targeted by donor mitochondria displaying either IgG1 or anti-CD31 antibodies two hours after transplantation. Endothelial cells are stained by anti-CD31 antibodies (red). All cells are stained with Phalloidin dye (green). Donor mitochondria displaying antibodies are stained by anti-SNAP antibodies (magenta). CD31-positive cells are outlined with grey dashed lines. l. Quantification of the efficacy of the delivery of antibody-displaying mitochondria two hours after transplantation. n = 4, P < 0.0001 (left) and P = 0.0158 (right), two-sided Welch’s t test. m. Immunostaining of tdTomato-expressing blood vessel organoids for CD31 (cyan), PDGFRβ (magenta), and RFP (red). The presence of vascular organoid cell types was validated in at least three induction batches. * P < 0.05, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m. Scale bars, 10 µm (c, d, e), 50 µm (h, k, m). The diagrams in b and j were created using BioRender; Ayupov, T. https://BioRender.com/fv9sxoi (2026).

Article Snippet: Human cardiac fibroblasts were obtained from LifeLine Cell Technology (FC-0060) and were maintained in FibroLife S2 Fibroblast Medium (Complete Kit, LifeLine Cell Technology, LL-0011) supplemented with 1% penicillin–streptomycin (Gibco, 15140-122).

Techniques: Conjugation Assay, Expressing, Membrane, Staining, Immunostaining, Isolation, Control, Western Blot, Incubation, Transplantation Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Cardiomyocyte-derived TGFB3 attenuates cardiac fibrosis and preserves cardiac function in heart failure

doi: 10.1038/s41598-026-42367-5

Figure Lengend Snippet: TGFB3 upregulation predominantly occurs in cardiomyocytes under pathological conditions. (A) UMAP visualization of single-nucleus RNA sequencing (snRNA-seq) data from mouse hearts (dataset SCP1303, Single Cell Portal). Left: expression pattern of Tgfb3 across all cardiac cell populations, with color intensity representing normalized expression levels. Right: cells colored according to cluster identity. (B) Bar graph showing Tgfb3 expression levels in cardiomyocytes (CM), fibroblasts (FB), and endothelial cells (EC) from sham and TAC groups, derived from a publicly available transcriptomic dataset (GEO accession: GSE180720 ). (C) qPCR analysis of Tgfb3 expression in isolated cardiomyocyte (CM) and non-cardiomyocyte (non-CM) fractions ( n = 4 per group). (D) Immunoblot analysis of TGFB3 protein levels in CM and non-CM fractions. cTnT and αSMA were used as markers of CM and non-CM, respectively; HSP90 served as a loading control. (E) qPCR analysis of Tgfb3 expression in primary cardiomyocytes treated with AngII (5 µM) or vehicle for 24 h ( n = 3 per group). (F) qPCR analysis of Tgfb3 expression in HL-1 cells treated with AngII (5 µM) or vehicle for 24 h ( n = 3 per group). (G) Immunoblot analysis of TGFB3 protein levels in HL-1 cells treated with AngII (5 µM) or vehicle for 48 h; HSP90 served as a loading control. Data are presented as mean ± SEM from three independent experiments. Statistical significance was tested by two-tailed unpaired Student’s t test in ( B, C, E, F ). p-values are indicated above each comparison.

Article Snippet: HEK293T cells (ATCC, CRL-3216) and primary cardiac fibroblasts were maintained in complete medium consisting of high-glucose Dulbecco’s Modified Eagle Medium (DMEM; BasalMedia, L110KJ) supplemented with 10% fetal bovine serum (FBS; Sigma, F8318) and 1% penicillin–streptomycin (BasalMedia, S110JV).

Techniques: RNA Sequencing, Single Cell, Expressing, Derivative Assay, Isolation, Western Blot, Control, Two Tailed Test, Comparison

Journal: Scientific Reports

Article Title: Cardiomyocyte-derived TGFB3 attenuates cardiac fibrosis and preserves cardiac function in heart failure

doi: 10.1038/s41598-026-42367-5

Figure Lengend Snippet: Transcriptomic and mechanistic analysis of fibrosis-related pathways regulated by TGFB3. (A) Volcano plot showing differentially expressed genes in heart tissue from myocardium-specific Tgfb3 knockout (Tgfb3^ΔMyh6) versus control (Tgfb3^fl/fl) mice. (B) Reactome pathway enrichment analysis of upregulated genes from RNA-seq of Tgfb3^ΔMyh6 hearts. (C) Venn diagram and heatmap illustrating five fibrosis-related genes that are highly expressed in the hearts of Tgfb3^ΔMyh6 mice. (D) qPCR analysis of Serpinf1 and Ctgf expression in heart from Tgfb3^fl/fl and Tgfb3^ΔMyh6 mice ( n = 8 per group). (E) Immunoblot analysis of SERPINF1 and CTGF protein expression in heart from Tgfb3^fl/fl and Tgfb3^ΔMyh6 mice. HSP90 was used as a loading control. (F) Representative immunofluorescence images showing the localization of TGFB3 (green), CTGF (cyan), p-SMAD3 (red), and nuclei stained with DAPI (blue) in heart from Tgfb3^fl/fl and Tgfb3^ΔMyh6 mice. Scale bar, 50 μm. (G) qPCR analysis of Acta2, Ctgf and Serpine1 expression in primary cardiac fibroblasts (cFB) treated with vehicle, TGF-β1 (5 ng/mL), and/or TGF-β3 (5 ng/mL) for 24 h ( n = 4 per group). (H) Immunoblot analysis of CTGF and SERPINE1 protein expression in primary cardiac fibroblasts (cFB) treated with Veh, TGF-β1 (5ng/ml) and/or TGF-β3 (5ng/ml) for 48 h. HSP90 serving as a loading control. (I) Immunoblot analysis of p-SMAD3, SMAD3, p-SMAD2 and SMAD2 protein expression in primary cardiac fibroblasts (cFB) treated with Veh, TGF-β1 (5ng/ml) and/or TGF-β3 (5ng/ml) for 15 min. HSP90 serving as a loading control. (J) Luciferase assay results of HEK293T stimulated by Veh, TGF-β1 (5ng/ml) and/or TGF-β3 (5ng/ml) for 48 h. pCAGA12-luc plasmids were used for transfection. (K) Western blot analysis demonstrates co-immunoprecipitation of TGFB1, TGFB3 and TGFBR2 in primary cardiac analysis treated with recombinant TGFB1 and TGFB3. Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t-test ( D, G) and one-way ANOVA by Tukey’s multiple comparisons test ( J ). p-values are indicated above each comparison.

Article Snippet: HEK293T cells (ATCC, CRL-3216) and primary cardiac fibroblasts were maintained in complete medium consisting of high-glucose Dulbecco’s Modified Eagle Medium (DMEM; BasalMedia, L110KJ) supplemented with 10% fetal bovine serum (FBS; Sigma, F8318) and 1% penicillin–streptomycin (BasalMedia, S110JV).

Techniques: Knock-Out, Control, RNA Sequencing, Expressing, Western Blot, Immunofluorescence, Staining, Luciferase, Transfection, Immunoprecipitation, Recombinant, Two Tailed Test, Comparison