Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles

Figures

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  Figure 1. TEM immunogold analysis for Cx26 in dH9c2 cells. (A) Low magnification of representative cell counterstained with uranyl acetate and lead citrate without immunogold treatment. Scale bar 4 µm. (B–D) Representative images of immunoreaction (arrows) at level of different cell compartments, rough endoplasmic reticulum (RER) and Golgi complexes (GC). Scale bars 200 nm. N, nucleus; PM, plasma membrane.

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  Figure 2. TEM immunogold analysis for Cx26 in dH9c2 cytoplasmic vesicles. (A,B) Arrows point to gold particles on different sized vesicles (V). (C–E) Representative images of multivesicular bodies (MVB) showing immunoreactivity. Scale bars 200 nm (A,C–E), 100 nm (B). RER, rough endoplasmic reticulum.

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  Figure 3. TEM double immunogold analysis for Cx26 (10 nm gold particles) and flotillin or CD63 (20 nm gold particles) on cytoplasmic vesicles. (A–C) Representative images of dH9c2 cells showing double immunoreaction for Cx26 and flotillin (A,B) and for Cx26 and CD63 (C) on multivesicular bodies (MVB). (D–F) Heart sections showing double immunoreactivity for Cx26 and flotillin (D,E) and for Cx26 and CD63 (F) on small vesicles (arrows) sometimes associated with sarcomeres (E,F). Scale bars 200 nm (A,B,D–F), 100 nm (C). V, vesicles.

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  Figure 4. TEM immunogold analysis for Cx26 and Cx43 in cardiomyocytes of heart tissue. (A–C) Cx26 immunogold analysis. (A) Lateral plasma membrane (LPM) of two adjacent cardiomyocytes showing scarce amount of gold particles (arrow). Scale bar 200 nm. (B,C) Representative images showing no immunoreaction on intercalated discs (arrows) as well as on gap junctions (GJ). Scale bars 100 nm. (D,E) Cx43 immunogold analysis. Arrows point to gold particles mainly at level of intercalated discs. Scale bars 200 nm. M, mitochondrion; MY, myofibrils; SR sarcoplasmic reticulum.

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  Figure 5. TEM immunogold analysis for Cx26 in delivery and degradation of subcellular structures. (A) Cardiomyocytes of heart tissue: representative image of an annular gap junction (AGJ), associated with a mitochondrion (M), showing immunogold reaction on (arrows) and inside the membrane. Scale bar 200 nm. (B,C) Representative images of autophagic vacuoles showing scattered gold particles (arrows) in dH9c2 cells. Scale bars 600 nm (B), 200 nm (C).

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  Figure 6. TEM immunogold analysis for Cx26 in non-canonical subcellular structures. Representative images of immunopositivity (arrows) in nucleus (N) of dH9c2 cells (A) and heart tissue cardiomyocytes (B). (C–E) Representative images of heart tissue cardiomyocytes evidencing immunogold particles on the inner membrane and cristae of mitochondria (M), on myofibrils (MY) and sarcoplasmic reticulum (SR). (F) Representative images of immunogold particles on myofibrils (MY) of dH9c2 cells. Scale bars 200 nm (A,C–F), 500 nm (B).

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  Figure 7. Negative staining of EVs from dH9c2 supernatant and Cx26 immunolocalization. (A) Representative images of EVs having a roundish and cup-shaped (squares) morphology. Scale bar 100 nm. (B) Diagram showing the EVs relative distribution in different dimension classes. The total number of counted EVs was 429. (C–E) Representative images of Cx26 immunogold localization performed with 20 nm (C,D) and 10 nm (E) gold particles conjugated anti rabbit secondary antibodies. Scale bars 100 nm.

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  Figure 8. Negative controls for TEM immunogold analysis. Negative controls were obtained by omitting the primary antibody (A–D) or by using RαCx32 as primary antibody (E). Representative images of dH9c2 cells (A–C) and cardiomyocyte sarcoplasm of heart sections (D,E) showing no immunoreaction. Scale bars 200 nm (A,C–E), 500 nm (B). M, mitochondrion; MY, myofibrils; N, nucleus; SR, sarcoplasmic reticulum; V, vesicle.