human cardiac fibroblasts  (Innoprot Inc)

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