human desmoglein-2 fc chimera protein (dsg2  (R&D Systems)

Journal: Basic Research in Cardiology

Article Title: The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner

doi: 10.1007/s00395-020-0805-3

Figure Lengend Snippet: Digitoxin increases DSG2 binding force. AFM force measurements were performed with a DSG2-coated tip on living HL-1 cardiac myocytes at areas of cell–cell contact as shown in a , green rectangle marked with an asterisk. These scanning regions were selected from AFM topography images (example in right panel) generated on confluent cell layers as confirmed by bright-field microscopy (example in left panel). Green dotted lines exemplarily highlight boundaries of two cells in both images. Scale bar: 10 µm. Force measurements were performed before and after incubation with digitoxin 1 µM for 30–90 min. N = 7 independent experiments and coatings. Bars indicate mean values ± SD. b Representative AFM topography and binding maps depicting the distribution of DSG2-mediated binding events along the cell–cell border of HL-1 cells. Each white pixel indicates one binding event, scale bar: 1 µm. Green dotted line highlights cell–cell border. c Corresponding distribution ratio of DSG2 binding events is defined as binding frequency at the cell border versus the adjacent cell area. To calculate the distribution ratio, a 10-pixel-wide region at cell–cell contacts was defined as cell border as indicated in b . Every dot indicates one independent experiment. Two-tailed, paired Student’s t test, (ns) P > 0.05 vs. control. d Corresponding mean number of DSG2 binding events per force map. Every dot indicates one independent experiment. Two-tailed, paired Student’s t test, (ns) P > 0.05 vs. control. e Corresponding scatter blot of binding forces of all binding events. Every dot indicates the force value of one binding event. Mann-Whitney test, *P < 0.05

Article Snippet: Briefly, eukaryotically expressed DSG2-Fc protein was coupled to Si3N4 AFM cantilevers (MLCT probes, Bruker, Calle Tecate, CA, USA) in a concentration of 0.15 mg/ml via a bifunctional polyethylene glycol spacer (acetal-PEG-NHS, Gruber Lab, Institute of Biophysics, Linz, Austria).

Techniques: Binding Assay, Generated, Microscopy, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Basic Research in Cardiology

Article Title: The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner

doi: 10.1007/s00395-020-0805-3

Figure Lengend Snippet: Digitoxin causes accumulation of DSG2, DP and PG at the cell–cell contacts. a Representative immunostaining images of HL-1 cardiac myocytes treated with digitoxin at indicated concentrations for 60 min and stained for DP (in merge: red), DSG2 (in merge: green), PG, or NCAD, scale bar: 20 µm. For better visibility, single channel images were inverted. N = 6 independent experiments with two dependent replicates per experiment. b Representative immunostaining images of ICDs of murine cardiac slices treated with digitoxin 1 µM for 60 min and stained for DP (in merge: green), DSG2 (in merge: red), scale bar 5 µm. For better visibility, single channel images were inverted. N = 3 mice per condition. Analysis of signal intensity of DP or DSG2 at the ICD. Every dot corresponds to one ICD, 20 ICDs per mouse measured, mean ± SD. Two-way ANOVA with Sidak’s post-hoc test. *P < 0.05, (ns) P > 0.05

Article Snippet: Briefly, eukaryotically expressed DSG2-Fc protein was coupled to Si3N4 AFM cantilevers (MLCT probes, Bruker, Calle Tecate, CA, USA) in a concentration of 0.15 mg/ml via a bifunctional polyethylene glycol spacer (acetal-PEG-NHS, Gruber Lab, Institute of Biophysics, Linz, Austria).

Techniques: Immunostaining, Staining

Journal: Basic Research in Cardiology

Article Title: The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner

doi: 10.1007/s00395-020-0805-3

Figure Lengend Snippet: Effect of digitoxin is dependent on desmosomal proteins. a Representative Western blot analysis of indicated ICD proteins in HL-1 cells treated with digitoxin at concentrations of 100 nM or 1 µM for 60 min. α-Tubulin served as loading control. N = 6 independent experiments. b Triton-X-extraction assay to separate cytoskeletal and non-cytoskeletal-bound fractions of HL-1 cardiac myocytes treated with digitoxin at concentrations of 100 nM or 1 µM for 60 min. GAPDH served for the non-cytoskeletal and desmin for the cytoskeletal-bound fraction as loading and separation control. Values above the lanes depict the mean band intensity by densitometric quantification compared to the respective loading control as fold of control ± SD, N = 5 independent experiments. Kruskal–Wallis with Dunn’s post-hoc test. *P < 0.05, (ns) P > 0.05. c Dissociation assay of HL-1 cells transfected with non-targeting siRNA (siNT) or siRNAs to reduce protein levels of PG (siPG), DSG2 (siDSG2), or DP (siDP) and treated with digitoxin 1 µM for 60 min. Bars indicate mean value ± SD. Every dot represents the mean value of two to three dependent replicates. Two-way ANOVA with Sidak’s post-hoc test, *P < 0.05. N = 15 (siNT), 7 (siDSG2, siDP) or 6 (siPG) independent experiments, respectively

Article Snippet: Briefly, eukaryotically expressed DSG2-Fc protein was coupled to Si3N4 AFM cantilevers (MLCT probes, Bruker, Calle Tecate, CA, USA) in a concentration of 0.15 mg/ml via a bifunctional polyethylene glycol spacer (acetal-PEG-NHS, Gruber Lab, Institute of Biophysics, Linz, Austria).

Techniques: Western Blot, Transfection

Journal: Basic Research in Cardiology

Article Title: The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner

doi: 10.1007/s00395-020-0805-3

Figure Lengend Snippet: Effect of digitoxin is dependent on phosphorylation of ERK1/2. a Western blot analysis of HL-1 cells treated with digitoxin 100 nM or 1 µM for 60 min to reveal phosphorylation state of ERK1/2, p38MAPK (p38) and Src. α-Tubulin served as loading control. Values above the lanes depict the mean band intensity by densitometric quantification compared to the respective total protein as fold of control ± SD, N = 6 independent experiments. Kruskal–Wallis with Dunn’s post-hoc test. *P < 0.05. b Dissociation assay of HL-1 cells pre-treated with 10 µM UO 126 for 60 min to inhibit ERK1/2 phosphorylation with subsequent application of 1 µM digitoxin for 60 min. Bars indicate mean value ± SD. Every dot represents the mean value of two to four dependent replicates. Two-way ANOVA with Sidak’s post-hoc test, *P < 0.05, (ns) P > 0.05. N = 9 (control) or 6 (UO 126 10 µM) independent experiments, respectively. c Representative immunostaining images of DSG2 (in merge: green) and DP (in merge: red) in HL-1 cardiac myocytes pre-treated with UO 126 10 µM for 60 min with subsequent application of digitoxin 1 µM for 60 min; scale bar: 20 µm. For better visibility, single channel images were inverted. N = 6 independent experiments with two dependent replicates per experiment

Article Snippet: Briefly, eukaryotically expressed DSG2-Fc protein was coupled to Si3N4 AFM cantilevers (MLCT probes, Bruker, Calle Tecate, CA, USA) in a concentration of 0.15 mg/ml via a bifunctional polyethylene glycol spacer (acetal-PEG-NHS, Gruber Lab, Institute of Biophysics, Linz, Austria).

Techniques: Western Blot, Immunostaining

Journal: Basic Research in Cardiology

Article Title: The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner

doi: 10.1007/s00395-020-0805-3

Figure Lengend Snippet: Digitoxin and adrenergic signaling induces ERK1/2 activation without additive effects. a Western blot analysis of HL-1 cells treated with digitoxin 1 µM or F / R 5 µM/10 µM for 60 min to reveal phosphorylation state of ERK1/2. Bar graphs depict the mean band intensity by densitometric quantification compared to the respective loading control as fold of control ± SD, N = 6 independent experiments. Kruskal–Wallis with Dunn’s post-hoc test. *P < 0.05 vs. control. b Dissociation assay of HL-1 cells treated with digitoxin 1 µM, FR 5 µM/10 µM or combination of both for 60 min. Bars indicate mean value ± SD. Every dot represents the mean value of two to three dependent replicates. Two-way ANOVA with Tukey’s post-hoc test, *P < 0.05 vs control, # P < 0.05, (ns) P > 0.05. N = 7 (control), 6 (digitoxin 1 µM) or 5 ( F / R , F / R + digitoxin 1 µM) independent experiments, respectively. c Representative immunostaining images of DSG2 (in merge: red) and DP (in merge: green) in HL-1 cardiac myocytes treated with digitoxin 1 µM, F / R 5 µM/10 µM or combination of both for 60 min. Scale bar: 10 µm. For better visibility, single channel images were inverted. N = 4 independent experiments with two dependent replicates per experiment. Western blot analysis of HL-1 cells ( d ) or murine cardiac slices ( e ) treated with digitoxin 1 µM or F / R 5 µM/ 10 µM for 60 min to reveal phosphorylation state of PG at S665. Bar graphs depict the mean band intensity by densitometric quantification compared to the total protein as fold of control ± SD, N = 6 (control, digitoxin 1 µM, F / R + digitoxin 1 µM) or 5 ( F / R ) independent experiments, respectively in d . N = 5 mouse hearts per genotype in e . Kruskal–Wallis with Dunn’s post-hoc test. *P < 0.05, (ns) P > 0.05. f Representative immunostaining images of DSG2 (in merge: red) in HL-1 cardiac myocytes overexpressing GFP-tagged (in merge: green) PG phospho-deficient at S665 (PG-S665A-GFP) or wild-type PG (PG-WT-GFP) as control. Cells were treated with digitoxin 1 µM for 60 min. Scale bar: 10 µm. For better visibility, single channel images were inverted. N = 3 independent experiments with two dependent replicates per experiment

Article Snippet: Briefly, eukaryotically expressed DSG2-Fc protein was coupled to Si3N4 AFM cantilevers (MLCT probes, Bruker, Calle Tecate, CA, USA) in a concentration of 0.15 mg/ml via a bifunctional polyethylene glycol spacer (acetal-PEG-NHS, Gruber Lab, Institute of Biophysics, Linz, Austria).

Techniques: Activation Assay, Western Blot, Immunostaining