jasplakinolide thermofisher  (Thermo Fisher)

Journal: Communications Biology

Article Title: Sustained strain applied at high rates drives dynamic tensioning in epithelial cells

doi: 10.1038/s42003-025-08210-9

Figure Lengend Snippet: a Time series of F-actin in cells stretched to 50% strain at 50%s −1 and 0.5%s −1 . Scale bar: 10 μm. b Relative change in F-actin intensity (i) and circular variance (CV) of F-actin fibers over time (ii) while the strain was kept constant within the confinement region. Scale bar: 10 μm. c Relative change in F-actin intensity (i) and the angle of fibers relative to the stretching direction over time within the gap region (90° is parallel to stretching direction) near the cell-cell junction while the strain was held constant. Scale bar: 10 μm. d The evolution of stress over time in control ( N = 10, orange) and Jasplakinolide (Jas) treated ( N = 13, brown) cell pairs stretched at 50%s −1 – 125% strain (average of all curves for each condition ± s.e). e The average of the B vs C values calculated from fitting individual curves for the Jas treated cell pairs and the control condition on a semi logarithmic scale. Inset shows the clustered B vs C values where solid triangles and hollow circles represent tensioned and relaxed responses, respectively. f The probability of tensioning for the control and Jas treated cell pairs in response to 125% strain applied at 50%s −1 . g The evolution of stress over time in control ( N = 40, green), Bleb ( N = 10, olive green), Y-27632 ( N = 7, purple), and Lat B ( N = 16, pink) treated cell pairs stretched at 25%s −1 to 50% (average of all curves for each condition ± s.e.). h The corresponding B vs C values calculated and clustered for the data presented in ( g ). i The probability of tensioning for the cell pairs treated with actomyosin modulators and the control condition in response to 50% strain applied at 25%s −1 .

Article Snippet: For latrunculin B (Abcam) treatment, LatB was diluted in DMSO and then diluted in DMEM to a final concentration of 200 nM and exposed to the cells for 30 min. For blebbistatin (Sigma Aldrich) treatment, stock bleb was diluted in DMSO and then diluted in DMEM to final concentrations of 3.4 and 4.25 μM and exposed to cells for 2 h. For Y27632 (Abcam) treatment, stock Y27632 was diluted in DMSO and then diluted in DMEM to a final concentration of 30 μM and exposed to cells for 10 min. For Jasplakinolide (Thermofisher) treatment, stock Jasplakinolide was diluted in DMSO and then diluted in DMEM to a final concentration of 0.1 μM and exposed cells for 10 min. For all drug treatments the media containing the drug was replaced with fresh media before beginning the stretching experiment.

Techniques: Control

Journal: Pflugers Archiv

Article Title: SECS, drugs, and Rac1&Rho: regulation of EnNaC in vascular endothelial cells

doi: 10.1007/s00424-025-03093-5

Figure Lengend Snippet: Rapid EnNaC membrane insertion and altered nanomechanics after aldosterone stimulation. A Time-series of 10 nM aldosterone application on EA.hy926 cells and quantification of EnNaC membrane abundance via Q-dot-based fluorescence staining. Already after 2 min of 10 nM aldosterone stimulation, a significant increase of the number of EnNaC within the membrane could be observed, reaching the highest channel density after 6 min ( N = 3, n = 60; **** p < 0.0001 vs. control (0 min), Kruskal–Wallis test). B EnNaC density was analysed in a concentration series with 7 min stimulation in EA.hy926 cells, already showing an increase of EnNaC membrane abundance after stimulation with 0.1 nM aldosterone ( N = 3, n = 60; **** p < 0.0001 vs. control (0 nM), Kruskal–Wallis test). C Short-term application (7 min) of 1 nM aldosterone was combined with the MR-antagonist spironolactone (100 nM), and EnNaC membrane abundance was analysed using EA.hy926 cells. Aldosterone increased the number of EnNaC within the membrane, whereas spironolactone did not additionally affect the EnNaC number ( N = 3, n = 60; **** p < 0.0001 vs. control). D Functional EnNaC inhibition acutely changed cortical stiffness of EA.hy926 cells (stimulation < 2 min) after treatment with amiloride as well as benzamil measured by AFM-based nanoindentation ( N = 3, n = 59–71; ** p < 0.01, **** p < 0.0001 vs. control, Kruskal–Wallis test). E Stabilization of filamentous F-actin by Jasplakinolide abolished the Amiloride-induced softening of the actin cortex (EA.hy926, N = 5, n = 25–129, * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. control, ANOVA). F F-actin stabilization by Jasplakinolide (JAS) directly affected the number of EnNaC channels in the membrane (HUVECs, N = 4, n = 80, *** p < 0,001, Mann–Whitney test). G Alterations of F- to G-actin ratio in response to EnNaC inhibition were analysed in a western blot-based approach in EA.hy926. Amiloride and benzamil both led to a reduction of F-actin to G-actin proportion ( N = 3, ns. vs. control). H Actin polymerization was analysed by phalloidin-TRITC fluorescence intensity analysis. Amiloride, benzamil, and the positive control, cytochalasin D (CytD), led to a significant reduction of phalloidin fluorescence (EA.hy926, N = 3, n = 60; * p < 0.05, **** p < 0.0001 vs. control, Kruskal–Wallis test)

Article Snippet: In vitro cell culture stimulation: After reaching confluence, cells were treated with the following substances, depending on the experiments: 0.1–10 nM aldosterone, 100 nM spironolactone, 1 μM amiloride, 0.1 μM benzamil, 50 nM cytochalasin D (CytD), 10 μM CK548 (all from Sigma Aldrich, St. Louis, MO, USA), 10 μM LY294002, 0.5 μM jasplakinolide (Thermo Fisher Scientific), 10 μM chelerythrin chloride, 5 μg/mL brefeldin A (both Biomol, Hamburg, Germany), 50 μM NSC23766 (Tocris, Bristol, UK), 1 μg/mL CT04 (Cytoskeleton, Denver, CO, USA), and 30 μM Pitstop-2 (Abcam, Cambridge, UK).

Techniques: Membrane, Fluorescence, Staining, Control, Concentration Assay, Functional Assay, Inhibition, MANN-WHITNEY, Western Blot, Positive Control