Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: Ensheathment and Cx43 plaque in expanded human epidermis. Magnification with filled arrowhead indicating part of an IENF, labeled via PGP9.5 (magenta), tunneling through a keratinocyte, labeled via phalloidin (grey). Additional hollow arrowhead showing a Cx43 plaque in contact with IENF. Physical 1.2 µm stack step size, translating to 276 nm biological step size. Framerate of two planes per second. Expansion factor corrected scale bar 1 µm. Abbreviations: Cx43, connexin 43; IENF, intraepidermal nerve fiber; PGP9.5, protein gene product-9.5.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques:

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Principle of unbiased correlation using intrinsic landmarks. IF channels are locked and hidden behind nuclear DAPI signal, which is used as only visible fluorescent channel, applied as landmark channel (blue). DAPI signal and SEM nucleus texture are used to achieve unbiased correlation of IF and SEM information. After optimization of the correlation, the channels of interest are made visible, overlaid and regions of interest selected. No further changes of image locations were applied afterwards. IF signal specificity. ( B ) Four consecutive tissue sections with nuclear DAPI (blue), DP-1 (yellow), and PGP9.5 (magenta) labeling showing traceable persistent labeling. Arrowhead indicates PGP9.5 positive nerve fiber and numbers show position of respective nuclei. Nucleus number 2 is ceasing within the z-stack. Scale bar 5 µm. ( C ) Application of secondary antibodies Al488-anti mouse (green), SeTau-647-anti rabbit (yellow), and Cy3-anti guinea pig (red) alone showed no fluorescent signal. DAPI signal (blue) for orientation. Scale bar 10 µm. Abbreviations: Al488, Alexa Fluor 488; Cy3, cyanine 3; DAPI, 4',6-diamidino-2-phenylindole; DP-1, desmoplakin 1; IF, immunofluorescence, PGP9.5, protein product 9.5 SEM, scanning electron microscopy.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Tissue, Labeling, Immunofluorescence, Scanning Electron Microscopy

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Tracking of Cx43 plaques in epidermal layers. First panel illustrates SEM overview of epidermal layers corresponding to five consecutive sections of IF images showing Cx43 signal (red) and nuclei (blue). Asterisks mark examples of traced Cx43 plaques. ( B ) Cx43 plaques at keratinocyte-nerve fiber close contact sites. Nerve fibers processing between keratinocytes in skin samples of two male subjects (M1, M2) and one female subject (F1). Each row represents four consecutive sections of 100 nm thickness. First tile shows keratinocyte cell bodies (orange), nuclei (blue), and nerve fibers (magenta) in pseudo color with Cx43 signal (red). Correlated PGP9.5 labeling (magenta) locates at nerve fibers and Cx43 labeling (yellow) indicates Cx43 plaques. Insets show magnification of contact area. Scale bars: 5 µm ( A ), 1 µm ( B ), magnified insets: 500 nm. Abbreviations: Cx43, connexin 43; IF, Immunofluorescence; PGP9.5, protein gene product-9.5; SEM, scanning electron microscopy.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Labeling, Immunofluorescence, Scanning Electron Microscopy

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Overview of skin section prior to expansion with PGP9.5-labeled IENF (magenta), Cx43 (yellow), and nuclear DAPI (blue); dotted line illustrates epidermis-dermis border and arrow indicates IENF. White rectangle marks inset enlarged in B. ( B ) Enlarged area prior to expansion and ( C ) matched area post-expansion at same magnification with addition of actin marker phalloidin (grey); white rectangles mark inset area of D and E. ( D ) Enlarged IENF area at ×20 magnification and ( E ) at ×63 magnification with arrowheads indicating Cx43 plaque at IENF. Inset in E marks enlarged area in E`-E```. ( E` ) shows z-plane prior to E, ( E`` ) same z-plane as E, and E``` z-plane step after E. White scale bars indicate pre-expansion state, yellow scale bars are corrected for expansion factor. Scale bars: 50 µm ( A ), 5 µm ( B, C ), 2 µm ( D, E ), 500 nm ( E`-E``` ). Z-step size of 1.2 µm translates to approximate 274 nm in expanded gel. Abbreviations: Cx43, connexin 43; IENF, intraepidermal nerve fiber; PGP9.5, protein gene product-9.5.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Labeling, Marker, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Workflow for ensheathment and Cx43 quantification (see also ). ( B ) Magnitude of skin tissue expansion across investigated sections via comparison of epidermal nuclei. Median nucleus size between samples pre-ExM (8.73±0.54 µm) and post ExM (32.46±3.54 µm) denotes a median ExM factor of 3.83±0.36. ( C ) Extracted fiber ensheathment ratio (PGP9.5-positive px without WGA pixel co-localization divided by total PGP9.5 px count) in HC versus SFN patients. ( C ) Extracted fiber ratio (total PGP9.5 px versus PGP9.5 non WGA overlap px) in HC versus SFN patients (n=13 HC and 10 HC ROIs from n=5 HC and n=4 SFN samples; p=0.41; shown as box & whiskers plot with minimum and maximum). ( D ) Cx43 contact ratio (PGP9.5 px with Cx43-WGA double positive px divided by total PGP9.5 px) in HC versus SFN (n=13 HC and 10 SFN ROIs from n=5 HC and n=4 SFN samples; p=0.34; shown as box & whiskers plot with minimum and maximum). Expansion factor corrected scale bars (yellow) of exemplary segmentation: 5 µm. See also . Abbreviations: HC, healthy control; Cx43, connexin 43; NCU, neuro-cutaneous unit; PGP9.5, protein gene product 9.5; px, pixel; SFN, small fiber neuropathy; WGA, wheat germ agglutinin. Figure 5—source data 1. Descriptive data and values for nucleus based calculation of expansion factors. Figure 5—source data 2. Statistical details and input values for .

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Tissue, Comparison, Control, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Confocal overview image of Ctx-positive sensory neurites (green), Cx43 positive (orange) keratinocytes with membrane labeling of keratinocytes via WGA (magenta) and nuclear DAPI (blue). ( B ) Inset of ensheathment area from A. ( C ) Single plane lattice SIM image and respective inset area with 3D visualization of z-stack (2.925 µm depth, 0.196 µm steps) showing nucleus, Cx43, and neurite signal ( C` ) and including WGA ( C`` ). ( D ) Confocal overview image of Ctx-positive sensory neurites (green), Cx43-positive (orange) keratinocytes with membrane labeling of keratinocytes via WGA (magenta), and nuclei (blue). Arrowheads indicate Cx43 - neurite contact areas. ( E ) Single plane lattice SIM image and respective inset area with 3D visualization, numbers represent single Cx43 plaques ( F ) of z-stack (2.925 µm depth, 0.196 µm steps). ( F`-F`` `) detailed neurite - Cx43 contact areas. Co-culture kept in keratinocyte medium. Scale bars: 20 µm ( A, D ), 5 µm ( C-C``, E, F`-F```` ), 1 µm ( F ). Abbreviations: Ctx, cholera toxin subunit B; Cx43, connexin 43; DAPI, 4',6-diamidino-2-phenylindole; SIM, structured illumination microscopy; WGA, wheat germ agglutinin.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Confocal, Membrane, Labeling, Co-culture Assay, Microscopy, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: Primary and secondary antibodies and marker with specifications.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Marker

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet:

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques:

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet:

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Immunofluorescence

Journal: European Heart Journal

Article Title: Left bundle branch vs biventricular pacing: mechanistic insights from a canine model

doi: 10.1093/eurheartj/ehaf1093

Figure Lengend Snippet: Molecular targets in cardiac remodeling by BiVP and LBBP. Western blotting results of BNP, TGF-β, KCNIP2, SERCA2a, Cx43, ANKRD1 protein expression (A, C, E) in septum and LV lateral walls from NC, DHF, and BiVP and LBBP groups, and relative protein expression (B, D, F). Represented images and statistical data (G) of cardiac fibrosis detected using Masson's staining. † P < .05 between septal and lateral walls. Abbreviations as in

Article Snippet: The levels of several key proteins were examined using western blot, including ANKRD1 (Proteintech, 11427-1-AP), BNP (Abclonal, A23996), Cx43 (Proteintech, 26980-1-AP), GSNOR (Proteintech, 66193-1-Ig), GLUD1 (Proteintech, 14299-1-AP), KCNIP2 (Abclonal, A7100), SERCA2a (Abclonal, A0098), TGF-β (Proteintech, 81746-2-RR), and GAPDH (Proteintech, 10494-1-AP).

Techniques: Western Blot, Expressing, Staining

Journal: European Heart Journal

Article Title: Left bundle branch vs biventricular pacing: mechanistic insights from a canine model

doi: 10.1093/eurheartj/ehaf1093

Figure Lengend Snippet: Overview of the key findings. This study compares the treatment efficacy of LBBP and BiVP, and relating electrocardiographic, echocardiographic and cellular (reverse) remodeling in a canine DHF model. The results show that LBBP restores electrical synchrony (QRSd) to a similar extent as BiVP, while leads to larger LV-GLS improvement than BiVP. Both LBBP and BiVP reverse the protein level of KCNIP2, SERCA2a and Cx43. Moreover, LBBP reverses abnormalities in cytoskeleton-associated proteins, TGF-β signaling, and SERCA2a expression more than BiVP, as well as enhances myocardial energy metabolism via more efficient TCA cycling and ATP production. ANKRD1, ankyrin repeat domain 1; ATP, adenosine triphosphate; BiVP, biventricular pacing; BNP, B-type natriuretic peptide; Cx43, connexin 43; DHF dyssynchronous heart failure; ECG, electrocardiogram; echo, echocardiogram; GLUD1, glutamate dehydrogenase 1; GSNOR S-nitrosoglutathione reductase; KCNIP2, potassium voltage-gated channel interacting protein 2; LBBP, left bundle branch pacing; LV-GLS, left ventricular global longitudinal strain; NC, normal control; SERCA2a, sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a; TCA, tricarboxylic acid; TGF-β, Transforming growth factor-β.

Article Snippet: The levels of several key proteins were examined using western blot, including ANKRD1 (Proteintech, 11427-1-AP), BNP (Abclonal, A23996), Cx43 (Proteintech, 26980-1-AP), GSNOR (Proteintech, 66193-1-Ig), GLUD1 (Proteintech, 14299-1-AP), KCNIP2 (Abclonal, A7100), SERCA2a (Abclonal, A0098), TGF-β (Proteintech, 81746-2-RR), and GAPDH (Proteintech, 10494-1-AP).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

doi: 10.64898/2026.02.08.704470

Figure Lengend Snippet: (A) Confocal immunocytochemical image of fixed cells stained to visualize endogenous Cx43 (green) and nuclei (blue). (B) Confocal live-cell image of cells transfected to express mEmerald-Cx43 (green) and the plasma membrane marker mCherry-CAAX (magenta). (C and C’) Confocal live image of cells transfected to express Halo-Cx43 (green), shown with (C) or without (C’) DIC overlay. (D and E) Transmission electron micrographs of a gap junction plaque (D) and an annular gap junction (E) in which the typical pentalaminar membrane morphology can be distinguished. In A-E, solid arrows represent gap junction plaques; dashed arrows represent the invaginated region of the gap junction plaque; arrowheads represent annular gap junctions. (F) Cells in various cell cycle stages were fixed and stained to visualize actin (magenta), Cx43 (green), and nuclei (blue). (F’) Enlargements of mitotic nanotubes in outlined areas in F to show association with Cx43-positive structures (arrows). (G) Cellular distribution of Cx43-positive structures across cell cycle stages. The bar graph represents the mean total area of Cx43 structures per compartment per cell ± SEM. Across 3 experiments, 79 cells were analyzed, with n ≥ 8 per cell cycle stage. (J) Micrograph of a membrane extension that forms a bridge between two cells. The extension is closed at the distal end (J’) where it makes contact with another cell. Contact sites shown in A (boxes J’ and J’’) were magnified to show the typical gap junction plaque membrane. (J’) Contact site between the closed end of the membrane extension and the adjacent cell. (J’’) Contact site between an area along the length of the membrane extension and the tip of another membrane extension. Scale bars = 5µm (A, F, F’), 10µm (B, C, C’), 100nm (D, E), 2µm (J), 100nm (J’, J’’).

Article Snippet: Plasmids encoding the gap junction markers mEmerald-Cx43 (Addgene, Watertown, MA, plasmid #54055) and Halo-Cx43 were gifts from Michael Davidson and John O’Brien, respectively.

Techniques: Staining, Transfection, Clinical Proteomics, Membrane, Marker, Transmission Assay

Journal: bioRxiv

Article Title: Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

doi: 10.64898/2026.02.08.704470

Figure Lengend Snippet: (A) Time-lapse of gap junction plaque turnover in dividing and non-dividing cells. Gap junction plaques were monitored between pairs of interphase cells that later underwent mitosis (Dividing Cells) and pairs of interphase cells that did not divide over ≥ 16 hours of imaging (Nondividing Cells). Large plaques were present at cell-cell contacts at t=0 in both cell pair types (arrows). However, plaque internalization occurred more frequently, resulting in more annular gap junctions in the cytoplasm (arrowheads) of dividing cells than in non-dividing cells. In the montage shown, new annular gap junctions were similarly distributed between the cytoplasm of both cells (Cells 1 and 2). However, among the dividing cells, annular gap junctions were predominantly released into the cell that initially underwent mitosis, compared with the cell that divided later (Cell 4). After 80 minutes, little to no gap junction plaques remained at the surface of the dividing cells, whereas plaques between non-dividing cells showed no apparent change in size. (B) Time-lapse of gap junction dynamics as cells enter, undergo, and exit mitosis. Cells initially in interphase (t = 0:00) were observed to change shape, from flat to round (t = 9:20) and then divide to form daughter cells (t = 10:55) that flattened as they entered interphase (t = 14:40). New gap junction plaques were observed between daughter cells (t=14:40; also enlarged and shown with green channel only). (C) Gap junction plaque internalization into interphase cells in B. The gap junction plaque area has been enlarged, and the mEmerald channel (Cx43) is shown only. The mEmerald channel (Cx43) is shown only at 5-minute intervals for the first 1:20. Solid arrows follow the release of one annular gap junction over time, while dashed arrows follow the release and fission of a second annular gap junction. (D) Mitotic cell nanotubes from an area in B (t = 9:10) that has been enlarged to show Cx43-positive structures (arrows) visible along the mitotic nanotube as well as at both ends of the nanotube. (E) Mitotic nanotube changes shown with three-dimensional rotation of the images in A (t = 8:45 - 9:10). Note the change in Cx43-positive structure distribution within nanotubes over time and that the mitotic nanotubes remained above the substrate. Cells shown were synchronized, transfected to express mEmerald-Cx43 (green) and mCherry-CAAX (magenta), and imaged at 5-minute intervals. Scale bars = 5µm (A, C, D); 10µm (B, E).

Article Snippet: Plasmids encoding the gap junction markers mEmerald-Cx43 (Addgene, Watertown, MA, plasmid #54055) and Halo-Cx43 were gifts from Michael Davidson and John O’Brien, respectively.

Techniques: Imaging, Transfection

Journal: bioRxiv

Article Title: Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

doi: 10.64898/2026.02.08.704470

Figure Lengend Snippet: (A) Mitotic nanotubes in the area between a rounding mitotic cell and a neighboring cell. The solid arrow (0-34 min) represents a linear Cx43-positive structure within the mitotic nanotube. With time, the Cx43-positive structure elongates (0-26 min), fragments (30 min), and moves toward the dividing cell (34 min) before being lost from view (38 min). (B) Transfer of a Cx43-positive structure via mitotic nanotubes. A Cx43-positive structure (arrowhead), which resembles an annular gap junction, moved between two cells and changed shape within the mitotic nanotube before entering the cytoplasm. Some Cx43-positive material remained at one end of the mitotic nanotube (arrows). (C) Movement of a Cx43-positive structure along a mitotic nanotube. A mitotic-nanotube-associated Cx43 structure (arrows), initially at one end of the mitotic nanotube (0 min), moves along a straight path toward the other cell (1.5-10.5 min) and eventually enters the cytoplasm of the other cell (13.5 min). In this figure, cells were transfected to express mEmerald-Cx43 (green) and mCherry-CAAX (magenta) and confocal Z-stacks (step size = 0.25µm) were captured at 4-minute (A) or 1.5-minute intervals (B, C). Scale bars = 5µm (A, C); 2.5µm (B).

Article Snippet: Plasmids encoding the gap junction markers mEmerald-Cx43 (Addgene, Watertown, MA, plasmid #54055) and Halo-Cx43 were gifts from Michael Davidson and John O’Brien, respectively.

Techniques: Transfection